Methods and compositions for detecting rare cells from a biological sample

ABSTRACT

The present invention provides methods and compositions for isolating and detecting rare cells from a biological sample containing other types of cells. In particular, the present invention includes a debulking step that uses a microfabricated filters for filtering fluid samples and the enriched rare cells can be used in a downstream process such as identifies, characterizes or even grown in culture or used in other ways. The invention also include a method of determining the aggressiveness of the tumor or of the number or proportion of cancer cells in the enriched sample by detecting the presence or amount of telomerase activity or telomerase nucleic acid or telomerase expression after enrichment of rare cells. This invention further provides an efficient and rapid method to specifically remove red blood cells as well as white blood cells from a biological sample.

RELATED APPLICATIONS

This application claims benefit of priority to provisional applicationSer. No. 60/831,156, filed Jul. 14, 2006, and is a continuation in partof application Ser. No. 11/497,919, filed Aug. 2, 2006; each of thoseapplications is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of bioseparationand cell detection, and in particular to the field of biological sampleprocessing to detect rare cells, and downstream applications for thepurpose of screening for high risk population, diagnosing a disease,predicting disease or treatment outcome, monitoring a disease state orresponse to a therapy, optimizing a treatment regimen or developing anew therapy.

The mortality associated with malignant tumors is mostly due to theformation of metastasis in tissues and organs distant from the primarytumor. The early detection of the metastasis is a very importantdeterminant of the probability of survival for cancer patients(Feldstein M, Zelen M, Inferring the natural time history of breastcancer: implications for tumor growth rate and early detection. BreastCancer Res Treat. 1984; 4(1):3-10; Senie R T, Lesser M, Kinne D W, RosenP P, Method of tumor detection influences disease-free survival of womenwith breast carcinoma, Cancer. 1994 Mar. 15; 73(6):1666-72; Carlson J A,Slominski A, Linette G P, Mysliborski J, Hill J, Mihm M C Jr, Ross J S,Malignant melanoma 2003: predisposition, diagnosis, prognosis, andstaging. Am J Clin Pathol. 2003 December; 120 Suppl:S101-27).

Early detection of tumors and monitoring of tumor growth are considereda very critical element in the successful treatment of cancer patients.Current diagnostic technologies rely largely on imaging andhistopathology. Various imaging and scanning methods allow the detectionof tumor masses based on the differential metabolic activity or tissuedensity. Other imaging technologies such as colonoscopy and branchoscopyhelp identify tumor tissue by directly imaging through the surface of alumen. These methodologies have a lower limit of resolution of a fewmillimeters, which is equivalent to a mass already containing a largenumber of tumor cells (>2×10⁸). At this stage a tumor mass might becapable of shedding cancer cells in the bloodstream, with the potentialto originate metastasis. Histopathology allows the diagnosis of a tumorutilizing a tissue sample obtained by means of a biopsy. While thisapproach provides direct visualization of the tumor cells, it'sapplicability in the diagnosis or monitoring of a tumor can be limited.Often, the location of the tumor is such that obtaining a biopsy samplemay be impractical. In other cases, periodic monitoring by a temporalseries of biopsy cannot be preformed at high enough frequency to provideuseful information regarding the efficacy of a treatment and theprogression of the disease. Another important limitation of the currenthistopathological approaches is that they can only provide dataregarding the specific sites from which the biopsy was obtained,possibly overlooking the metastasis at other locations. Traditionalhistopathology can also be a risky process, during which tumor tissuesor cells may be carried out to contaminate distal locations, causingpotential metastasis during the biopsy procedure.

Typical progression of a localized tumor into a malignant cancer withmetastasis involves multiple steps. At relatively early stages the tumormass grows and the cells utilize nutrients available in the host tissueby simple diffusion. Later, once the tumor mass exceeds ˜1 mm indiameter, the tumor becomes vascularized, a step necessary for the tumorcells to have adequate supply of nutrients and retain their ability togrow. The vascularization step is driven in part by angiogenic factorsreleased by the tumor cells. As the tumor growth progresses, the cellslose more and more of the properties of the tissue of origin includingthe ability to tightly interact with the neighboring cells (see forexample Blanco M J, Moreno-Bueno G, Sarrio D, Locascio A, Cano A,Palacios J, Nieto M A, Correlation of Snail expression with histologicalgrade and lymph node status in breast carcinomas. Oncogene. 2002 May 9;21(20):3241-6). Eventually a small number of tumor cells will leak intothe blood vessels that are in direct contact with the tumor. Once incirculation, the circulating tumor cells can be carried to distant sitesin the body. The majority of these cells will not adapt to theenvironment of the bloodstream and will die while still in thecirculation. However, occasionally a subset of cells may survive in thecirculation for a longer time, by chance, or because of their moreresilient property. When a circulating tumor cell enters the capillariesin a distal region, it may remain trapped at that location. A series ofevents may then take place for these cells to attach and translocateacross the endothelial wall of the capillary. If the tumor cell survivesthe harsh environment in the blood stream until it crosses the capillarywall, it can invade the surrounding tissue and establish a metastasis atthe new location.

As a general rule, the more aggressive a cancer grows and the moremetastases it forms, the more difficult it will be to cure. Localizedtumors can be treated by surgical removal or by chemotherapy or acombination of the two. However, once the cancer cells have establishedmultiple colonies at different locations in the body, surgicalinterventions at many sites or on multiple organs in the body becomeimpractical and of limited therapeutic value. A cancer characterized bya single or multiple metastasis may be treated with chemotherapy.However, even in this case the success of the therapy may be limitedbecause the different cancer colonies (the primary tumor and themetastasis) often respond differently to any give chemotherapy treatment(El Hilali N, Rubio N, Blanco J. Different effect of paclitaxel onprimary tumor mass, tumor cell contents, and metastases for fourexperimental human prostate tumors expressing luciferase. Clin CancerRes. 2005 Feb. 1; 11(3):1253-8). The differential response to treatmentof the different cancer colonies is attributed to two main factors:genetic heterogeneity of the cancer cells at the different sites andlocal environmental factors differentially affecting cancer cellsurvival at the various locations.

All of these considerations emphasize the importance of detectingmetastasis at early stages. However, major challenges to early diagnosisare posed by the difficulty of detecting small metastases. A metastasiscan be established starting from a single cell shed off by a primarytumor. Current imaging methodologies (X-ray, PET-scan, CT-scan) cannotprovide early diagnosis of metastasis because their sensitivity in notsufficient to detect a single metastatic cell, but can only detect ametastasis once it grows to several millimeter in diameter and containsmore than 100 million cells. The ability to detect cancer cells incirculation early on during the establishment of a metastasis would beof great clinical relevance. Although a tumor mass can shed asignificant number of cells on a daily basis, the number of circulatingcancer cells in any given sample of blood of clinically relevant volume(5 to 40 mL) is very low. These cells are often present in the blood ata frequency of one cancer cell per 10⁷-10⁸ white blood cells. This makesit very difficult to isolate and detect circulating cancer cells earlyin the metastatic process.

Tumors can occur in different tissues, such as epithelial andmesenchymal tissues. The majority of human solid tumors originate fromepithelial cells that have undergone a transformation of their geneticmaterial and their phenotype and escape the checkpoints that keep cellgrowth and cell division under control. It is generally recognized thata single mutation is not sufficient to generate a cancer cell but,instead, a sequence of mutations is necessary to initiate a tumor. Thetumor cells are genetically unstable and continue to mutate and generatevariants throughout the entire progression of the disease. The highgenetic instability is the source of the heterogeneity observed amongthe tumor cells. In turn, the varied cell population in the tumorbecomes the stage for evolutionary competition and selection in whichthe cells with the faster division time, highest metabolic rate andlowest differentiation grade, tend to grow and divide faster andeventually take over the entire population. Since new mutations and newtumor cell variants are constantly generated, a high degree of diversityis maintained in the tumor cell population.

The genetic instability and heterogeneity in the cells of the primarytumor has no clinical consequences since surgical intervention canremove the entire tumor mass and all of its cell variants. However, oncethe disease reaches the stage of metastatic cancer, the geneticdiversity of the cancer cells in the primary tumor and the metastasisposes a major challenge for both the diagnosis as well as the treatmentof the metastasis.

Epithelial cells are tightly bound to one another and form sheets(called epithelia), which line all the cavities and surfaces of thebody. One of the distinctive features of epithelial cells is theformation of tight intercellular junctions mediated by specializedprotein complexes expressed on the surface of the epithelial cells.These cell-cell interactions leave very little space for extracellularspace in epithelia and render the epithelia a selective barrier to thepassage and diffusion of water, solutes and cells from one compartmentof the body to another.

During the transformation of an epithelial cell into a metastatic cancercell, numerous dramatic changes in the biological properties of the celltake place. The rate of growth and cell division is increased and thecell becomes progressively less dependent upon the growth control signalpresent in the normal tissue. Most importantly, in order to becomemetastatic, the cell needs to lose the key distinctive feature ofepithelial cells: its ability to tightly bind to neighboring cells. In awell-known example of this situation, in breast cancer, the epithelialcell adhesion molecule E-Cadherin is lost in the cancer cells thatprogress to the metastatic stage. The protein E-Cadherin is actuallyknown to be a “tumor suppressor”, that is, a factor whose higherexpression tends to suppress the progression of cancer. The changesassociated with the transformation of epithelial cells into tumor cellsare so dramatic that the cancer cells cannot be identified as epithelialcells anymore. This transformation is often referred-to asepithelial-mesenchymal transition, to indicate the conversion of anepithelial cell, characterized by strong interactions with neighborscells, into a mesenchymal cell, a cell with loose or no interaction withother cells and free to migrate in a tissue.

In recent years it has also became apparent that the metastases arelikely originating from a small subpopulation of tumor cells calledcancer stem cells. These cells may be present in the tumor mass at afrequency <2% and are less differentiated than the majority of the cellsin the tumor. Cancer stem cells have gained the property of duplicatingthemselves in addition to producing a differentiated offspring cell,which allow them to become “immortal”, or having unlimited selfreproduction potential. These cells have a high proliferation rate andcan easily establish new metastasis at sites distant from the primarytumor. The importance of cancer stem cells in the diagnosis andtreatment of metastatic cancer has been made clear by the observationthat often the remission, or response to therapy as measured clinicallyusing imaging of the primary tumor mass, is not predictive of thepatient survival or time to recurrence. This apparent paradox can beexplained by recognizing that the treatment capable (for example) ofreducing the tumor mass, may be ineffective on the cancer stem cells,which are the main reason for uncontrolled and unlimited proliferation,and could still form distal metastasis.

At present, accumulating reports indicate that circulating tumor cells(CTCs) may be found in patients even before the primary tumor isdetected. In addition to a potential role in early diagnosis andprognostication, CTCs may play a major role in characterizing geneticand immunophenotypic changes with tumor progression, thereby helping toguide individualized therapy. Though various techniques have beenapplied to isolate and characterize CTCs, many of them share the similarprinciple, i.e. antibody based positive selection. Apparently,application of this strategy for CTCs detection is limited by theavailability, sensitivity, and specificity of antibodies againstbiomarkers on different tumor cells. Alternative strategies involvenegative depletion of red blood cells (RBCs) and white blood cells(RBCs). The filtration approach to depletion the smaller RBCs and WBCsbased on size risks losing target cells since certain target cells suchas some CTCs can be as small as WBCs. Other approaches involving thelysis of RBCs might risk damaging target cells.

There is a need for a diagnostic methodology which has high sensitivity,and is capable of detecting cancer cells or cancer stem cells, or acertain population of more aggressive forms of cancer cells, such ascancer stem cells, in the body of a patient when said patient still hasa relatively small tumor mass. In addition there is a need for atechnology that can isolate and identify cancer cells or a certainpopulation of more aggressive forms of cancer cells, such as cancer stemcells, in the body, regardless of the stage of the cancer, and the levelof transformation accumulated by the cancer cells. Such a technologywould allow for the identification of metastatic cancer disease at anearly stage, or provide means for monitoring disease progression,response to therapy, or status of disease relapse. The present inventionprovides these and other benefits.

BRIEF SUMMARY OF THE INVENTION

The present invention recognizes that screening, diagnosis, prognosis,and treatment of many conditions can depend on the enrichment of rarecells from a complex fluid sample. Often, enrichment can be accomplishedby one or more separation steps. In particular, the present inventionrecognizes that the enrichment or separation of rare cells includingmalignant cells from patient samples, such as the isolation of cancerouscells from patient body fluid samples, can aid in the detection andtyping of such malignant cells and therefore aid in diagnosticdecisions, as well as in the development of therapeutic modalities forpatients.

The present methods utilize a negative or depletion approach forisolating rare cells from a sample. In these methods, a sample isprogressively enriched for the rare cells of interest by a series ofsteps that remove other components from the sample with highspecificity. Target cells, such as circulating tumor cells in bloodsamples, mesenchymal cells, epithelial cells, stem cells, mutated cells,and the like are then more easily identified in the enriched sample, andmay be isolated, quantified, further characterized, or even grown inculture or used in other ways. Because this approach relies on depletionof non-target cells, it overcomes disadvantages of many ‘positiveselection’ approaches for isolating rare cells. Those approaches canmiss the rare cells if such cells have mutated, for example, and nolonger express an expected surface antigen that is used to capture orlabel the target cells. Since mutations are commonly seen in cells thatsuch methods would desirably detect, such as cancer cells, the depletionmethods of the present invention have substantial advantages over‘positive selection’ methods for isolating rare cells from complexbiological samples.

A first aspect of the present invention is a method of enriching targetcells from a biological sample such as a peripheral blood sample, thoughother biological samples can also be similarly enriched. Target cellsmay be epithelial cells, mesenchymal cells, cancer cells, infectedcells, mutated cells, damaged cells, stem cells, or other cells thatoccur infrequently in biological samples. Often, the target cell is acell type whose presence, number, proportion, or properties are usefulfor a diagnostic or prognostic assessment of the subject from whom thebiological sample was collected.

In one aspect, the invention provides a method for isolating a targetcell from a biological sample containing other types of cells, saidmethod comprising:

-   -   a) enriching the sample by selectively removing at least one        non-target cell type without removing the target cell;    -   b) debulking the sample by reducing the sample volume without        removing the target cell; and    -   c) subjecting the enriched sample comprising the target cell to        a downstream process that identifies, characterizes, or utilizes        the target cell or information about its presence, proportion,        properties. In some embodiments, this method does not utilize        centrifugation.

In one aspect of the invention, the presence, number, proportion, orcharacteristics of the target cell(s), if present, can be used toprovide diagnostic or prognostic information about the subject from whomthe sample was collected. This may be done, for example, by correlationof the presence, number, proportion or properties of such target cellsin the subject to those parameters in samples from healthy and diseasedindividuals. The methods may be used to determine the presence or statusof cancer, for example, or to determine the subject's remission status,the effectiveness of a course of therapy, the metastatic potential ofthe cancer, and the like. It may be used to evaluate experimentaltherapies or drug candidates, too. In some embodiments, the methodincludes additional steps such as identification of the tissue origin ofthe target cell, which may assist in determining the location of a tumoror a metastasis site, or further steps such as growing a culture ofcells from the target cell(s) in a suitable medium. A culture of suchtarget cells can also be used to evaluate experimental therapies or drugcandidates.

In certain embodiments, after washing a blood sample, the blood sampleis centrifuged at a speed that enhances the recovery of a rare cell typeof interest, against serum proteins and blood constituents that havelower gravitational density than cells. In one embodiment of thisaspect, a blood sample (obtained from a cancer patient or from a subjectpotentially at risk for cancer) is washed and centrifuged at a speedthat enhances recovery of cancer cells.

In some embodiments of this method, during washing of a blood sample,the cells in the blood sample are concentrated to enhance their recoveryby means of a French press type of device. In one embodiment of thisaspect, a blood sample (obtained from a cancer patient) is washed byapplying a pressure differential on a filtration device, which allowsfor the fluid component of the blood sample to enter a separatecompartment from the one in which the cells are confined. This devicemay be a microfiltration device that selectively separates at least somefluid from a sample that contains target cells by passing the fluidthrough a filter having pores that are sized to permit some componentsof the sample to pass through and to substantially prevent passage ofthe target cells. In some embodiments, this filter is adapted to repelthe target cells from its surface by surface modifications of the filteritself, or by use of a force that selectively repels the target cellsfrom the filter, such as a dielectrophoretic force generated by one ormore electrodes on or near the filter.

In some embodiments, this method includes at least some of the followingsteps: providing a blood sample from a cancer patient; washing thesample by adding an artificial buffer followed by centrifugation; inwhich after the wash centrifugation, a wash supernatant and a washedpellet are obtained; resuspending the washed pellet, which represents anenriched sample; lysing certain non-target cells in the sample, such asred blood cells by adding an artificial buffer, and centrifuging toobtain a further enriched pellet containing the target cells and somenon-target cells and a supernatant containing lysed non-target cellresidues. Optionally, the lysing step can be repeated to achieve thebest result. Depending upon the sample characteristics, these steps andothers described below may be performed in any order, and may berepeated or omitted as needed to enrich the target cell population to asufficient degree for its intended use.

In certain embodiments, the present invention uses a specific bindingmember such as an antibody or molecule that specifically binds a surfacemolecule or moiety on a type of non-target cell (such as a WBC or otherhematopoietic cell) with lesser binding to the target cells as part ofthe enrichment process. Non-limiting examples of suitable specificbinding members include an antibody or molecule that binds CD3, CD11b,CD14, CD17, CD31, CD34, CD45, CD50, CD53, CD63, CD69, CD81, CD84, CD102,CD166, CD233, CD235, and CD236, or a combination of antibodies chosenfrom this list. The antibody may be utilized to remove white blood cellsfrom a blood sample, for example, as part of the enrichment process. Theantibody can optionally be bound to a solid support, which support maybe specially adapted to facilitate its removal from the sample. Inpreferred embodiments of the present invention, the antibody can be usedto remove white blood cells from a blood sample in a procedure forenriching cancer cells from a blood sample.

In one aspect, the invention provides methods for enriching a samplewith rare cells, using a French press type device. It also providesdevices to facilitate this method, which comprise a filter that ispreferably a rigid material having one or more pores through it, andoptionally having one or more electrodes on or adjacent to its surfaceto produce a dielectrophoretic effect that can repel cells in the samplefrom the surface of the filter and thereby improve the operation of thefilter.

Another aspect of the present invention is a method of enriching targetcells from a biological sample, which includes the following steps: a)removing white blood cells (WBCs) from said blood sample withmicroparticles and b) removing red blood cells (RBCs) from said bloodsample to enrich non-hematopoietic cells with a density-based approache.g., a ficoll gradient centrifugation, and c) performing an analysis,manipulation or application step with the enriched sample. Steps a) andb) can be performed in either order or at the same time.

In some embodiments, the present invention includes methods ofseparating sample components from a fluid sample using magneticparticles which could be any solid phase particle/microspheres such asmagnetic particle, sepharose, sephadex, and agarose etc based particles,and the particles are chemically modified to conjugate antibody or anyother proteins for target selection and binding.

In certain embodiments, the present invention involves the method ofremoval of WBCs from blood sample, which involves microparticles with adimension between 10 nm and 10 um, and linked to specific binding membersuch as an antibody or molecule that specifically binds to an antibodywhich recognizes antigens expressing on leukocyte, such as a CD45 and/orCD50 antibody and/or another leukocyte-specific antibody. In someembodiments, one specific binding member is used. In other embodiments,two or more different specific binding members are used.

In another embodiment of the present invention, the density-basedapproach involves centrifugation and usage of sugar or derivatives ofsugar such as ficoll as the material for establishing a densitygradient. Furthermore, the density media used in this method has thesame separation effect as ficoll diluted to 50%˜100% with a buffer.

In a certain embodiment, this invention is designed to very rapidlyremove plasma proteins, most WBCs and RBCs with lesser undesiredeffects, resulting in easy detection of target cells from remainingenriched cells. In addition, the current invention can also be appliedto separate plasma protein, enrich other rare cells, including stemcells, fetal cells, immune cells, etc, followed by downstream analysis,manipulations, and applications such as flowcytometry, PCR,immunofluorescence, immunocytochemistry, image analysis, enzymaticassays, gene expression profiling analysis, efficacy tests oftherapeutics, culturing of enriched rare cells, and therapeutic use ofenriched rare cells.

In some embodiment of the present invention, enriched cells are stainedwith both fluorescence-based reagents, and absorbance-based reagents,such as Hemotoxylin. Immunofluorescence and immunocytochemistryapproaches can be used at the same time to increase the number of stainsand biomarkers that can be employed to characterize the enriched sample.

In one embodiment, before applying any enrichment protocol described inthe present invention, a pre-labeled or non-labeled known cell type canbe mixed with the sample to be analyzed, with a specific labeling thatwill not be used for the identification or characterization of thetarget cells. This pre-mixed cell can serve as an “internal control”that qualifies the performance of the assay.

In some embodiments, the current invention can employ a multiplicity ofspecific binding members against a multiplicity of biomarkers on morethan one type of target cells, i.e., circulating tumor cells, andcirculating endothelial cells. Such procedures can enable thesimultaneous detection of two or more target cells, with differentpathophysiological significance or indications.

In a certain embodiment, target cells enriched by the current invention,are analyzed by fluorescence microscopy. To aid the accurate reading offluorescence-labeled samples, a slide or cover slide is prepared withsuitable methods such as laser, wet etch, dry etch, ion beam, etc, tocreate grid lines to assist the slide reading. The natural fluorescenceof the grid lines can be further enhanced by treating with fluorescentmaterials, such as quantum dots, or fluorescent dyes.

In one embodiment, the present invention is directed to a method fordetecting a non-hematopoietic cancer cell, in a blood sample, whichmethod comprises: a) providing a blood sample; b) removing red bloodcells (RBCs) from said blood sample, with a density-based approach,such, e.g., a Ficoll density centrifugation, and removing white bloodcells (WBCs) from said blood sample to provide an enriched samplecontaining the non-hematopoietic cell, e.g., a non-hematopoietic tumorcell, if any is present, from said blood sample, with amicroparticle-based approach; and c) assessing the presence, absenceand/or amount of said enriched non-hematopoietic cell or tumor cell. Themicroparticle-based approach often uses a particle that is 10 nm to 10microns in size, with a specific binding member linked to the particle.The specific binding member is typically an antibody or similar binderthat is specific for at least one surface antigen on leukocytes. Themicroparticle may comprise a magnetic material to facilitate separationof the particle from the enriched sample. Detection of the presence ornumber of such non-hematopoietic cancer cells can be used to assess thepresence, status, or progression of a non-hematopoietic cancer in thesubject from which the blood sample was taken.

A further aspect of the present invention is a method for identifyingcancer cells following their enrichment from a blood sample thatcomprises: enriching the cancer cells in a sample by removing themajority of the non-cancer cells from the sample, which may be a bloodsample, for example; specifically labeling the cancer cells with adetectable label after at least one such enrichment step; and countingthe number of labeled cancer cells in the enriched sample.Alternatively, the ratio or proportion of such enriched cancer cells toother cell types such as non-cancerous epithelial cells, or non-stemtype cancer cells, may be used as a diagnostic or prognostic indicatorthat may be more reliable than counting of the cancer cells. Thelabeling of the cancer cells is sometimes achieved using a plurality ofmarkers in order to obviate to the genetic instability and populationheterogeneity typical of cancer cells.

In a preferred embodiment, tumor cells are labeled and identified bybinding to a specific binding member or a multiplicity of specificbinding members recognizing one or more ligands selected from the groupconsisting of ACPP, AFP, albumin, ALCAM, AMAS, ARF6, ARMCX3, ATP1A1,BAG1, BJ-TSA-9, blc-2 βHCG, CA125, CA15-3, CA19-9, Cathepsin B1, CD44,CD44v6, CD56, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD147, CDH2,CDK4I, CDKN2A, CDX2, CEA, CLDN3, CLDN4, CLDN5, c-met, CST3,Cytokeratins, CK18, CK19, CK20, Desmoplakin-3, EAG1, EGFR, EGP2, EMA,ErbB2, ESR1, FAK, FOXA2, GalNac-T, GCTFTI5, GFAP, Haptoglobin-α, HCA,hCASK, HE4, HEPA1, hERG, HIP-1, HMB45, HSPA2, IGFR, IVL, KCNK-9,KHDRBS3, Ki67, Kv1.3, LAMB2, Lewis-Y antigen, LIMA, LMO6, LUNX, MAGE-3,MAGE-A3, mammoglobin, Maspin, Melan-A, MITF, MPP5, MPST, MUC-1, MUC5AC,NCAM-1, NSDHL, Oct4, OTC, p53, p97, p1B, PCNA, PGR, PMSA, PS-2, PSA,RPS6KA5, S100, S100A1, S100A2, S100B, SLC2A1, Smoothelin, SP-1, SPARC,Surfactant, Telomerase, TFAP2A, TITF1 (TTF1), TFF2, TRAIL, TRIM28,TRPM-8, TYR, Tyrosinase, TYRP1, Ubiquitin thiolesterase, VEGF, WT1,X-protein, ZNF165. Leading references for selected markers: Clin CancerRes. 2005 May 15; 11(10):3678-85 (CA15-3); Clin Cancer Res. 2004 Apr.15; 10(8):2812-7 (FAK); J Clin Invest. 2002 August; 110(3):351-60(HIP-1); Neoplasia. 2005 December; 7(12):1073-80 (BJ-TSA-9); CancerDetect Prev. 1994; 18(1):65-78 (AMAS); J Biol. Chem. 2006 Apr. 7;281(14):9719-27. Epub 2006 Jan. 27 (CD147); Lab Invest. 2003 September;83(9):1343-52 (TFF2); Cancer. 1995 Jun. 15; 75(12):2827-35 (Lewis Yantigen).

In the embodiments where a multiplicity of specific binding members isused, each binding member might carry none, the same, or differentlabels. In a preferred embodiment of the present invention the cancercells are identified with specific markers and also stained with markersof apoptosis. In some embodiments of the present invention the absolutenumber and/or the proportion of cancer cells which are also apoptoticcan be used as a predictor of the efficacy of an anticancer therapy orthe progression of the disease or the prognosis for the subject, orcombinations of the above.

The cancer cells isolated using the depletion methods described in thepresent invention are sometimes identified by their affinity for aspecific binding member, which is typically labeled for easy detection.Target cells may be recognized and labeled by their affinity forantibodies directed at one or more of the following: ACPP, AFP, albumin,ALCAM, AMAS, ARF6, ARMCX3, ATP1A1, BAG1, BJ-TSA-9, blc-2 βHCG, CAl25,CA15-3, CA19-9, Cathepsin B1, CD44, CD44v6, CD56, CD66a, CD66b, CD66c,CD66d, CD66e, CD66f, CD147, CDH2, CDK4I, CDKN2A, CDX2, CEA, CLDN3,CLDN4, CLDN5, c-met, CST3, Cytokeratins, CK18, CK19, CK20,Desmoplakin-3, EAG1, EGFR, EGP2, EMA, ErbB2, ESR1, FAK, FOXA2, GalNac-T,GCTFTI5, GFAP, Haptoglobin-α, HCA, hCASK, HE4, HEPA1, hERG, HIP-1,HMB45, HSPA2, IGFR, IVL, KCNK-9, KHDRBS3, Ki67, Kv1.3, LAMB2, Lewis-Yantigen, LIMA, LMO6, LUNX, MAGE-3, MAGE-A3, mammoglobin, Maspin,Melan-A, MITF, MPP5, MPST, MUC-1, MUC5AC, NCAM-1, NSDHL, Oct4, OTC, p53,p97, p1B, PCNA, PGR, PMSA, PS-2, PSA, RPS6KA5, S100, S100A1, S100A2,S100B, SLC2A1, Smoothelin, SP-1, SPARC, Surfactant, Telomerase, TFAP2A,TITF1 (TTF1), TFF2, TRAIL, TRIM28, TRPM-8, TYR, Tyrosinase, TYRP1,Ubiquitin thiolesterase, VEGF, WT1, X-protein, ZNF165. In theembodiments where two or more specific binding members are used to labelthe target cells, each binding member might carry none, the same, ordifferent labels. The apoptotic cancer cells are often furtheridentified by detection of one or more of the following:Phosphatidylserine, DNA fragmentation, Cytochrome C, Caspase. The ratioof identified apoptotic cells to the overall specific rare cellpopulation identified by methods of the current invention may be used asa diagnostic index.

In some preferred embodiments, after debulking (reducing the volume orcell mass or both) of the sample and the removal of undesirablecomponents from the blood sample (e.g., hematopoietic cells,non-cancerous epithelial cells, etc.), labeled cancer cells are furtherenriched or isolated using magnetic capture methods, fluorescenceactivated cell sorting or laser cytometry. In some preferredembodiments, after debulking and the removal of various components fromthe sample to provide an enriched sample containing the cancer or othercells of interest, the rare cells of interest are labeled with aspecific marker. The labeled cancer cells are further analyzed usingspectral imaging, fluorescence microscopy, visible light microscopy, ormanual or automated image analysis.

In some preferred embodiments of the present invention the cancer cellsare identified using a combination of immunological and morphologicalcriteria. The cancer cells are frequently labeled by binding to aspecific binding member or a multiplicity of specific binding membersrecognizing one or more ligands selected from the group consisting ofACPP, AFP, albumin, ALCAM, AMAS, ARF6, ARMCX3, ATP1A1, BAG1, BJ-TSA-9,blc-2 βHCG, CA125, CA15-3, CA19-9, Cathepsin B1, CD44, CD44v6, CD56,CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD147, CDH2, CDK4I, CDKN2A,CDX2, CEA, CLDN3, CLDN4, CLDN5, c-met, CST3, Cytokeratins, CK18, CK19,CK20, Desmoplakin-3, EAG1, EGFR, EGP2, EMA, ErbB2, ESR1, FAK, FOXA2,GalNac-T, GCTFTI5, GFAP, Haptoglobin-α, HCA, hCASK, HE⁴, HEPA1, hERG,HIP-1, HMB45, HSPA2, IGFR, IVL, KCNK-9, KHDRBS3, Ki67, Kv1.3, LAMB2,Lewis-Y antigen, LIMA, LMO6, LUNX, MAGE-3, MAGE-A3, mammoglobin, Maspin,Melan-A, MITE, MPP5, MPST, MUC-1, MUC5AC, NCAM-1, NSDHL, Oct4, OTC, p53,p97, p1B, PCNA, PGR, PMSA, PS-2, PSA, RPS6KA5, S100, S100A1, S100A2,S100B, SLC2A1, Smoothelin, SP-1, SPARC, Surfactant, Telomerase, TFAP2A,TITF1 (TTF1), TFF2, TRAIL, TRIM28, TRPM-8, TYR, Tyrosinase, TYRP1,Ubiquitin thiolesterase, VEGF, WT1, X-protein, ZNF165. In theembodiments where a multiplicity of specific binding members is used,each binding member might carry none, the same, or different labels.Identification of cancer cells is often done by fluorescence microscopy,visible light microscopy, or manual or automated image analysis whereina cancer cell is identified as a cell labeled by binding membersrecognizing one, or two, or three or more of the markers listed above,and optionally also by specific morphological criteria related to thesize of its nucleus, shape and boundary characteristics of the nucleus,size of the whole cell, or the ratio of the cytoplasmic portion of thecells to its nucleus.

In another aspect, the present invention is directed to a method fordetecting a non-hematopoietic cell, e.g., a non-hematopoietic cancercell, in a blood sample, which method comprises steps including some orall of the following:

a) providing a blood sample;

b) removing red blood cells (RBCs) from said blood sample by selectivelylysing said RBCs, and removing the remaining hematopoietic cells such aswhite blood cells (WBCs) by specific binding of said hematopoietic cellsto specific binding members, such as binding WBCs to an anti-CD50antibody or another leukocyte-specific antibody, which may be linked toa solid surface such as a microparticle or a magnetic particle, toenrich the sample with the targeted cancer cells, if any are present;and

c) subjecting the enriched sample comprising the target cell to adownstream process that identifies, characterizes, or utilizes thetarget cell population, which can include assessing the presence,absence and/or amount of said enriched non-hematopoietic cell, e.g., anon-hematopoietic tumor cell, by: i) bright field microscopy followingnucleus and/or cytoplasm staining and applying morphological criteria,ii) immunostaining the enriched non-hematopoietic cell and counting thelabeled cells; iii) PCR-based analysis of the enriched non-hematopoieticcells; or detecting the presence or amount of an enzyme, surface markeror nucleic acid that is typically present in, and preferably is uniquelyassociated with, the target cell type.

In still another aspect, the present invention is directed to a kit fordetecting a non-hematopoietic cell, e.g., a non-hematopoietic tumorcell, in a blood sample, which kit comprises: a) means for removing redblood cells (RBCs) from a blood sample, with the proviso that said meansis not a Ficoll gradient centrifugation means; and means for removingwhite blood cells (WBCs) from said blood sample to enrich anon-hematopoietic cell, e.g., a non-hematopoietic tumor cell, if any,from said blood sample; and b) means for assessing the presence, absenceand/or amount of said enriched non-hematopoietic cell, e.g., anon-hematopoietic tumor cell, or means for assessing what fraction ofnon-hematopoietic cells are cancerous, or means for assessing whatfraction of target cells are apoptotic or possess stem cell traits.Suitable means for removing RBCs are disclosed herein and include alysis buffer or a solid surface or particle linked to a specific bindingmember that binds to RBCs. Suitable means for removing WBCs are alsodisclosed herein, and include, for example microparticles that areoptionally magnetic and are linked to at least one specific bindingmember that is selective for binding to leukocyte surface antigens.Means for assessing the presence, absence and/or amount of an enrichednon-hematopoietic cell are also disclosed herein, and include labeledantibodies directed against the tumor marker compounds described herein.

Another aspect of the present invention is the use of the presence ofcancer cells in a blood sample from a subject for the purpose ofdiagnosing cancer, wherein the presence of cancer cells in a bloodsample from said patient is indicative of the presence of a tumor in thebody of said subject, and may be indicative of a likelihood ofprogression or metastasis of the tumor. The cancer cells are isolatedusing one or more of the depletion methods described herein, or methodsknown in the art that can include positive selection methods, and thepositive identification of the cancer cells is obtained by labeling saidcells with one or more cancer marker-specific binders that recognize acancer marker selected from the following list: ACPP, AFP, albumin,ALCAM, AMAS, ARF6, ARMCX3, ATP1A1, BAG1, BJ-TSA-9, blc-2 βHCG, CA125,CA15-3, CA19-9, Cathepsin B1, CD44, CD44v6, CD56, CD66a, CD66b, CD66c,CD66d, CD66e, CD66f, CD147, CDH2, CDK4I, CDKN2A, CDX2, CEA, CLDN3,CLDN4, CLDN5, c-met, CST3, Cytokeratins, CK18, CK19, CK20,Desmoplakin-3, EAG1, EGFR, EGP2, EMA, ErbB2, ESR1, FAK, FOXA2, GalNac-T,GCTFTI5, GFAP, Haptoglobin-α, HCA, hCASK, HE4, HEPA1, hERG, HIP-1,HMB45, HSPA2, IGFR, WL, KCNK-9, KHDRBS3, Ki67, Kv1.3, LAMB2, Lewis-Yantigen, LIMA, LMO6, LUNX, MAGE-3, MAGE-A3, mammoglobin, Maspin,Melan-A, MITF, MPP5, MPST, MUC-1, MUC5AC, NCAM-1, NSDHL, Oct4, OTC, p53,p97, p1B, PCNA, PGR, PMSA, PS-2, PSA, RPS6KA5, S100, S100A1, S100A2,S100B, SLC2A1, Smoothelin, SP-1, SPARC, Surfactant, Telomerase, TFAP2A,TITF1 (TTF1), TFF2, TRAIL, TRIM28, TRPM-8, TYR, Tyrosinase, TYRP1,Ubiquitin thiolesterase, VEGF, WT1, X-protein, ZNF165. In embodimentswhere a multiplicity of specific binding members are used, each bindingmember might carry none, the same, or different labels.

Another aspect of the present invention is the monitoring of diseaseprogression, response to therapy, or relapse in a patient with cancer.During the natural progression of cancer disease, or as a result oftreatment, often cancer cells mutate their surface antigens, renderingpositive selection methods inadequate for detection of cancer cells inbiological samples. The depletion methods of the present invention areeffective in the isolation and identification of cancer cells in samplesfrom cancer patients. In a preferred embodiment of the invention, thenumber of cancer cells in a blood sample from a patient, as determinedby the methods of the present invention, is determined at different timeintervals during the progression of the disease or during a treatmentregimen lasting weeks, months or longer, or after a treatment regimenhas been discontinued. An increase in the number of cancer cells overtime is indicative of lack of response to therapy, relapse, higher riskof metastasis, worse prognosis, shortened projected survival time, orprogression to a higher stage of cancer or growth rate of the tumor, orcombinations of the above. A decrease or non-varying number of cancercells is indicative of favorable response to therapy, stable status ofthe disease, or shrinkage of the tumor or remission or combinations ofthe above.

In a preferred embodiment of the present invention, the cancer cellsisolated from a cancer patient are characterized for their nucleic acidcontent. This method can comprise steps such as: a) providing a bloodsample; b) removing red blood cells (RBCs) from said blood sample byselectively lysing said RBCs; c) removing white blood cells (WBCs) fromsaid blood sample by specific binding of said WBCs to an anti-CD50antibody or other leukocyte-specific antibody to enrich the sample withthe targeted cancer cells, if any are present; d) amplifying RNA and/orDNA from the isolated cancer cells; and e) characterizing the geneticcontent and/or gene expression pattern in the cancer cells using one ormore of the following methodologies: single nucleotide polymorphism,quantitative PCR, FISH, DNA sequencing, multiplexed PCR, determinationof the DNA methylation, total DNA content quantification, whole genomeamplification (WGA), CGH, laser dissection microscopy (LDM),amplification from RNA, Oligonucleotide ligation assay (OLA), Chromosomeimmuno-precipitation (CHIP), southern blot, hybridization,amplification, ligation, enzymatic assays.

Another aspect of the present invention is the monitoring of diseaseprogression, response to therapy, or relapse in a patient with cancer bymeans of assessment of the malignancy index. The malignancy index isdetermined as the ratio between a, the number of circulating candidatecancer cells or, circulating epithelial cells, and the number of cellse, which are non-apoptotic (or apoptotic) and/or over-express angiogenicmarkers and/or over-express proliferation markers and/or have reducedexpression of tumor suppression genes, and/or have lost markers ofdifferentiation and/or contains chromosomal deletion spanning tumorsuppressor genes and/or contain chromosomal amplification of tumorpromoters and/or over-express drug resistance factors and/or containspecific mutations. In certain embodiments, the α and ∈ values aredetermined by using markers that identify surface antigenscharacteristic of a particular cellular status, and the markers or setsof markers used to determine α and ∈ are not identical.

The number ∈ of epithelial cells or candidate cancer cells in a bloodsample can be determined as follows: a) providing a blood sample; b)removing red blood cells (RBCs) from said blood sample by selectivelylysing said RBCs, and removing white blood cells (WBCs) from said bloodsample by specific binding of said WBCs to an anti-CD50 antibody and/oranother leukocyte-specific antibody(s) to enrich the sample with thetargeted cancer cells, if any are present;

c) identification of the candidate cancer cells or the epithelial cellsin the enriched sample is achieved by labeling said cells with one ormore cancer markers selected from the following list: ACPP, AFP,albumin, ALCAM, AMAS, ARF6, ARMCX3, ATP1A1, BAG1, BJ-TSA-9, blc-2 βHCG,CA125, CA15-3, CA19-9, Cathepsin B1, CD44, CD44v6, CD56, CD66a, CD66b,CD66c, CD66d, CD66e, CD66f, CD147, CDH2, CDK4I, CDKN2A, CDX2, CEA,CLDN3, CLDN4, CLDN5, c-met, CST3, Cytokeratins, CK18, CK19, CK20,Desmoplakin-3, EAG1, EGFR, EGP2, EMA, ErbB2, ESR1, FAK, FOXA2, GalNac-T,GCTFTI5, GFAP, Haptoglobin-α, HCA, hCASK, HE4, HEPA1, hERG, HIP-1,HMB45, HSPA2, IGFR, IVL, KCNK-9, KHDRBS3, Ki67, Kv1.3, LAMB2, Lewis-Yantigen, LIMA, LMO6, LUNX, MAGE-3, MAGE-A3, mammoglobin, Maspin,Melan-A, MITF, MPP5, MPST, MUC-1, MUC5AC, NCAM-1, NSDHL, Oct4, OTC, p53,p97, p1B, PCNA, PGR, PMSA, PS-2, PSA, RPS6KA5, S100, S100A1, S100A2,S100B, SLC2A1, Smoothelin, SP-1, SPARC, Surfactant, Telomerase, TFAP2A,TITF1 (TTF1), TFF2, TRAIL, TRIM28, TRPM-8, TYR, Tyrosinase, TYRP1,Ubiquitin thiolesterase, VEGF, WT1, X-protein, ZNF165. In theembodiments where a multiplicity of specific binding members is used,each binding member might carry none, the same, or different labels.

The number α of cancer cells which are apoptotic, or highly malignant orde-differentiated or mutated or having high proliferation rate in ablood sample can be determined as follows: a) providing a blood sample;b) removing red blood cells (RBCs) from said blood sample by selectivelylysing said RBCs, and removing white blood cells (WBCs) from said bloodsample by specific binding of said WBCs to an anti-CD50 antibody and/oranother leukocyte-specific antibody(s) to enrich the sample with thetargeted cancer cells, if any are present; c) identifying the cancercells by labeling said cells with one or more cancer marker selectedfrom the following list: Phosphatidylserine, DNA fragmentation,Cytochrome C, Caspase expression, ACPP, AFP, albumin, ALCAM, AMAS, ARF6,ARMCX3, ATP1A1, BAG1, BJ-TSA-9, blc-2 βHCG, CA125, CA15-3, CA19-9,Cathepsin B1, CD44, CD44v6, CD56, CD66a, CD66b, CD66c, CD66d, CD66e,CD66f, CD147, CDH2, CDK4I, CDKN2A, CDX2, CEA, CLDN3, CLDN4, CLDN5,c-met, CST3, Cytokeratins, CK18, CK19, CK20, Desmoplakin-3, EAG1, EGFR,EGP2, EMA, ErbB2, ESR1, FAK, FOXA2, GalNac-T, GCTFTI5, GFAP,Haptoglobin-α, HCA, hCASK, HE4, HEPA1, hERG, HIP-1, HMB45, HSPA2, IGFR,IVL, KCNK-9, KHDRBS3, Ki67, Kv1.3, LAMB2, Lewis-Y antigen, LIMA, LMO6,LUNX, MAGE-3, MAGE-A3, mammoglobin, Maspin, Melan-A, MITF, MPP5, MPST,MUC-1, MUC5AC, NCAM-1, NSDHL, Oct4, OTC, p53, p97, p1B, PCNA, PGR, PMSA,PS-2, PSA, RPS6KA5, S100, S100A1, S100A2, S100B, SLC2A1, Smoothelin,SP-1, SPARC, Surfactant, Telomerase, TFAP2A, TITF1 (TTF1), TFF2, TRAIL,TRIM28, TRPM-8, TYR, Tyrosinase, TYRP1, Ubiquitin thiolesterase, VEGF,WT1, X-protein, ZNF165. In the embodiments where a multiplicity ofspecific binding members is used, each binding member might carry none,the same, or different labels.

In a preferred embodiment of the invention the malignancy index for apatient, as determined by the methods of the present invention, isdetermined at different time intervals before, during or aftertreatment. An increase in the malignancy index over time is indicativeof lack of response to therapy, higher risk of metastasis, worseprognosis, shortened projected survival time, or progression to a higherstage of cancer, growth of the tumor or combinations of the above. Adecrease or non-varying value of the malignancy index is indicative offavorable response to therapy, stable disease status or shrinkage of thetumor or remission or combinations of the above.

In a preferred embodiment of the present invention, the cancer cellsisolated from a cancer patient by either positive or negative(depletion) methods are characterized for their nucleic acid content.One such method comprises: a) providing a blood sample; b) removing redblood cells (RBCs) from said blood sample by selectively lysing saidRBCs, and removing white blood cells (WBCs) from said blood sample byspecific binding of said WBCs to an anti-CD50 antibody or anotherleukocyte-specific antibody to enrich the sample with the targetedcancer cells, if any are present; c) amplifying RNA and/or DNA from theisolated cancer cells; d) characterizing the genetic content and/or geneexpression pattern in the cancer cells using one or more of thefollowing methodologies: single nucleotide polymorphism, quantitativePCR, FISH, DNA sequencing, multiplexed PCR, determination of the DNAmethylation, total DNA content quantification, whole genomeamplification (WGA), CGH, LDM, amplification from RNA, OLA, CHIP,southern blot, hybridization, amplification, ligation, enzymatic assays.

In some preferred embodiments of the present invention the geneticcontent of the cancer cells is analyzed for the purpose of identifyingthe presence of mutations which can confer higher proliferation rates orchromosomal deletions spanning tumor suppressor genes or chromosomalamplification of tumor promoting genes.

In a further embodiment of the present invention, the characterizationof the genetic content of cancer cells isolated from a subject is usedto tailor a personalized therapeutic course specific to said subject orto the particular cancer phenotype.

Another aspect of the present invention includes the culturing and invitro propagation of cancer cells isolated from a biological sampleobtained from a cancer patient. This method can comprise: a) providing ablood sample; b) removing red blood cells (RBCs) from said blood sampleby selectively lysing said RBCs, and removing white blood cells (WBCs)from said blood sample by specific binding of said WBCs to an anti-CD50antibody or similar leukocyte-specific antibody to enrich the samplewith cancer cells; c) transferring of the enriched cancer cells sampleinto microtiter plates or similar container, and culturing in serum freemedia or media with serum concentration at or below 20% or preferablybelow about 5%.

In one aspect, the present invention provides a method to evaluateprogress in a clinical study of an experimental cancer therapy or drugcandidate. The subjects in such trials may be human, but frequently theywould include other mammals such as mice, rats, dogs, monkeys and thelike. In this aspect, the method can be used to provide a clinical endpoint to measure the efficacy of an experimental cancer therapy or drugcandidate that is faster and more quantitative than effectiveness,metastasis or recurrence data alone. This provides a more rapid andquantitative assessment of the efficacy of the therapeutic being tested,and it provides additional information about how the therapeutic affectsmetastasis and recurrence probabilities for the cancer being treated. Itthus provides more information about the overall effectiveness of theexperimental therapy and reduces the time required for such clinicaltrials.

In a preferred embodiment of the invention the cultured cancer cellsobtained from a cancer patient blood sample are used to test in vitrothe efficacy of candidate anti-cancer drugs or drug combinations beforeadministration of the drug(s) to said patient, or before decidingwhether to continue administration of the drug(s). In other embodiments,such cultured cancer cells are used to test new drug candidates or otherexperimental therapies as part of a clinical trial or even a primaryscreen for efficacy.

In another aspect of the present invention, the cancer cells isolatedfrom a cancer patient using the methods of the present invention, areimmortalized by in vitro culturing and selection which may or may not beaided by transfection of the cells with SV40 T-antigen or Telomerase orother suitable methods. The immortalized cells may then be used to testthe efficacy of anticancer agents, screen for new anticancer agents, orany other investigation requiring immortalized cell lines.

In another aspect of the present invention, the cancer cells isolatedfrom a cancer patient using the methods of the present invention areused in invasiveness assays.

Another aspect of the present invention is the use of the cancer cellsisolated from a cancer patient for the purpose of personalizedimmunotherapy, wherein proteins or nucleic acids or combinations thereofobtained from the cancer cells isolated from a cancer patient areincubated with WBC or a subfraction of WBC from said patient tostimulate a cancer specific immune response. The WBC or the subfractionof WBC exposed to the cancer cell tumor antigens are then re-inoculatedinto the patient.

In one embodiment of the present invention a blood sample from a cancerpatient is utilized for isolation of the cancer cells (from the cellularfraction) and performing standard biochemical assays (from the plasmafraction). After obtaining a blood sample from a cancer patient, saidsample is centrifuged to sediment all the cellular components. Followingthe centrifugation step, the plasma fraction or a portion of thisfraction is recovered and removed from the cell pellet. The cellularfraction is resuspended in an appropriate buffer and the cancer cellsare isolated using the depletion methods and cancer cell detectionmethods of the present invention. The plasma fraction is utilized forroutine clinical assays including, but not limited to, the determinationof plasma concentration of: Sodium, Potassium, Urea, Creatinine,Glucose, total protein, Albumin, Bilirubin, Alanine Transaminase,Alkaline Phosphatase, Gamma Glutamyl Transferase, Creatine Kinase,Aspartate Transaminase, Lactate Dehydrogenase, Amylase, C-reactiveprotein, D-dimer, Calcium, Copper, Zinc, Triglycerides, totalCholosterol, HDL Cholesterol, LDL Cholesterol, Alpha-fetoprotein,CA-125, Prostate specific antigen, TSH, FT4, FT3, ACTH, Cortisol,Prolactin, Testosterone.

In one embodiment of the present invention, following the isolation ofthe cancer cells from a blood sample using the depletion methodsdescribed in the present invention, the cancer cells can becharacterized using several immunoassays. For example, the cancer cellscan be lysed, and the lysate centrifuged, and subjected to ELISA assay.In this case, specific protein of interest expressed in the cancer cellscan be detected directly. This can provide a profile of the proteincontent in the cancer cells and allows the monitoring of how the cancercell phenotype changes during the course of the disease or during thetherapeutic treatment.

In some preferred embodiments of the present invention the cancer cells,following isolation, are characterized by one or more functional orenzymatic assays. Telomerase activity has been identified in lung cancercells as well as in cancer cells from many other cancers. Telomeraseactivity assay can be used to further characterize circulating tumorcells isolated with the depletion methods of the present invention orwith positive selection methods well known to those skilled in the arts.In this case, telomerase repeat amplification protocol (TRAP) can beperformed. Once the cancer cells are isolated, telomerase will beextracted using CHAPS based detergent buffer or any other suitablemethod. The supernatant of cell lysates will be used as template fortelomerase extension reaction by PCR. Fluorescent PCR products aregenerated using fluorescently labeled primers, followed by capillaryelectrophoresis measurements. The larger the amount of fluorescent PCRproduct generated or the larger the length of the telomerase repeatamplification products, the higher the telomerase activity of the cancercells in the sample, which can be an indicator of the aggressiveness ofthe tumor or of the number or fraction of cancer cells in the enrichedsample.

In recent years antibody-based therapy has had significant success inthe clinics and is now part of the standard arsenal used by cliniciansto fight cancer. The methods of the present invention provide a uniqueapproach in monitoring the effects and the efficacy of antibody-basedtherapies. In some embodiments the present invention can be used todetect the interaction between circulating cancer cells in the blood ofa cancer patient and an immunotherapeutic such as a humanized exogenousantibody used for the therapy. Whether isolated cancer cells are boundto a therapeutic antibody can be determined by isolating a cancer celland examining it for the presence of such antibodies; one such methodincludes: a) providing a blood sample; b) removing red blood cells(RBCs) from said blood sample by selectively lysing said RBCs, andremoving white blood cells (WBCs) from said blood sample by specificbinding of said WBCs to an anti-CD50 antibody or anotherleukocyte-specific antibody to enrich the sample with the targetedcancer cells, if any are present; c) identifying the cancer cells boundto the therapeutic antibody by labeling said cells with one or moreligands having specific affinity for the therapeutic antibody, andoptionally with additional binding members for the candidate cancercells; d) visualizing the labeled cells using the appropriate methods tovisualize a molecular ligand, which methods, include but are not limitedto fluorescence microscopy, immunohistochemistry, bright fieldmicroscopy and FACS, which methods are well known to those skilled inthe arts. However, this method can also be practiced in connection withpositive-selection cell isolation methods known in the art, such asthose that select the target cell from a biological fluid or sample byexposing the sample to a plurality of magnetic beads coated with aspecific binding member that binds to a surface marker on the targetcell.

In some preferred embodiments of the present invention the monitoring ofthe interaction between therapeutic antibody and the cancer cellspresent in a blood sample can be used to evaluate the patient's responseto therapy wherein when the fraction of cancer cells bound to animmunotherapeutic agent such as an antibody is above a predeterminedvalue, or increases over time for measurements at different time points,the outcome of the treatment is predicted to be favorable, and when thefraction of cancer cells bound to the antibody is below a predeterminedvalue, or decreases over time for measurements at different time points,the outcome of the treatment is predicted to be unfavorable. Where anabsolute number is not used, a ratio between the fraction of cells boundto the said antibody over the entire candidate cancer cell population orthe unbound candidate cancer cell fraction can also be used.

The enriched samples can be characterized and/or further manipulated ina downstream process performed on a miniaturized microfluidic device,such as a microfluidic chip or cartridge. Such device may comprise oneor more elements that use the principle(s) of dielectrophoresis, thermalgradient, acoustic, electroosmosis, or electromagnetic manipulation.Such device may also comprise filtration, mixing, sonication, thermalcycling, immunomagnetic separation, nucleic acid hybridization,two-photon microscopy, absorbance-based detection, fluorescence-baseddetection, FRET-based detection, immunorecognition, impedancemeasurement, electrical field stimulation, or cell culture functions.One preferred embodiment of the present invention involves obtaining asample enriched by a method described in the present invention, loadingthe enriched sample onto a microfluidic device that then separates rarecells of interest from the rest of the cells in the sample, directingthe separated cells to individual culture chambers for incubation,subjecting each culture chambers to therapeutic agents to be tested, andgenerating a suitable readout that is useful to evaluate the effects ofthe therapeutic agents.

The enrichment procedures described in the present invention may beminiaturized on a microfluidic device, such as a microchip or acartridge.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. French press device for cell sample debulking

A. Fluid sample containing target cells is shown inside the Frenchpress. (1) Main chamber containing the fluid samples to be debulked. (2)Filter. (3) Piston used to move the filter through the sample. (4) Inletvalve, to allow introducing of the sample in the main chamber (5) Outletvalve to allow exit of the debulked sample. (6) Target cell

B. Debulked sample showing new position of the filtration unit at theend of the debulking procedure

FIG. 2. Staining of human white blood cells with anti-CD50 monoclonalantibody

The results of CD50 staining on white blood cells are shown

FIG. 3. Staining of human cancer cells with anti-CD50 monoclonalantibody

Shows the lack of specific interaction between anti-CD50 antibody andcancer cells obtained from different types of cancers (indicated)

FIG. 4. Recovery of cancer cells from spiked blood samples

P, prostate cancer cells; L, lung cancer cells; B, breast cancer cells;C, Cervical cancer cells

FIG. 5. Cancer cells isolated from breast cancer patient

Top, normal cells from control healthy patient

Bottom three panels, examples of breast cancer cells.

FIG. 6. Changes of CTC count before and after chemotherapy (4-6 weeks)correlated with clinical response assessed by CT scan.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the manufacture procedures for devices and components aswell as the laboratory procedures described below are well known andcommonly employed in the art. Conventional methods are used for theseprocedures, such as those provided in the art and various generalreferences. Where a term is provided in the singular, the inventors alsocontemplate the plural of that term. As employed throughout thedisclosure, the following terms, unless otherwise indicated, shall beunderstood to have the following meanings: A “component” of a sample or“sample component” is any constituent of a sample, and can be an ion,molecule, compound, molecular complex, organelle, virus, cell,aggregate, or particle of any type, including colloids, aggregates,particulates, crystals, minerals, etc. A component of a sample can besoluble or insoluble in the sample media or a provided sample buffer orsample solution. A component of a sample can be in gaseous, liquid, orsolid form. A component of a sample may be a moiety or may not be amoiety.

A “moiety” or “moiety of interest” is any entity whose manipulation isdesirable. A moiety can be a solid, including a suspended solid, or canbe in soluble form. A moiety can be a molecule. Molecules that can bemanipulated include, but are not limited to, inorganic molecules,including ions and inorganic compounds, or can be organic molecules,including amino acids, peptides, proteins, glycoproteins, lipoproteins,glycolipoproteins, lipids, fats, sterols, sugars, carbohydrates, nucleicacid molecules, small organic molecules, or complex organic molecules. Amoiety can also be a molecular complex, can be an organelle, can be oneor more cells, including prokaryotic and eukaryotic cells, or can be oneor more etiological agents, including viruses, parasites, or prions, orportions thereof. A moiety can also be a crystal, mineral, colloid,fragment, micelle, droplet, bubble, or the like, and can comprise one ormore inorganic materials such as polymeric materials, metals, minerals,glass, ceramics, and the like. Moieties can also be aggregates ofmolecules, complexes, cells, organelles, viruses, etiological agents,crystals, colloids, or fragments. Cells can be any cells, includingprokaryotic and eukaryotic cells, whether living or dead. Eukaryoticcells can be of any type. Of particular interest are cells such as, butnot limited to, white blood cells, normal cells, modified cells, mutatedcells, malignant cells, stem cells, progenitor cells, fetal cells, andcells infected with an etiological agent, and bacterial cells. Moietiescan also be artificial particles such polystyrene microbeads, microbeadsof other polymer compositions, magnetic microbeads, and carbonmicrobeads.

As used herein, “manipulation” refers to moving or processing of themoieties, which results in one-, two- or three-dimensional movement ofthe moiety, whether within a single chamber or on a single chip, orbetween or among multiple chips and/or chambers. Moieties that aremanipulated by the methods of the present invention can optionally becoupled to binding partners, such as microparticles. Non-limitingexamples of the manipulations include transportation, capture, focusing,enrichment, concentration, aggregation, trapping, repulsion, levitation,separation, isolation or linear or other directed motion of themoieties. For effective manipulation of moieties coupled to bindingpartners, the binding partner and the physical force used in the methodmust be compatible. For example, binding partners with magneticproperties must be used with magnetic force. Similarly, binding partnerswith certain dielectric properties, e.g., plastic particles, polystyrenemicrobeads, must be used with dielectrophoretic force.

“Binding partner” or “binding member” refers to any substances that bothbind to the moieties with desired affinity or specificity and aremanipulatable with the desired physical force(s). Non-limiting examplesof the binding partners include cells, cellular organelles, viruses,microparticles or an aggregate or complex thereof, or an aggregate orcomplex of molecules.

A “microparticle” or “particle” is a structure of any shape and of anycomposition that is manipulatable by desired physical force(s). Themicroparticles used in the methods could have a dimension from about0.01 micron to about ten centimeters. Preferably, the microparticlesused in the methods have a dimension from about 0.1 micron to aboutseveral thousand microns. Frequently, they are in the range 0.1 to 10microns or 1-100 microns in size. Such particles or microparticles canbe comprised of any suitable material, such as glass or ceramics, and/orone or more polymers, such as, for example, nylon,polytetrafluoroethylene (TEFLON™), polystyrene, polyacrylamide,sepaharose, agarose, cellulose, cellulose derivatives, or dextran,and/or can comprise metals. Examples of microparticles include, but arenot limited to, plastic particles, ceramic particles, carbon particles,polystyrene microbeads, glass beads, magnetic beads, hollow glassspheres, metal particles, particles of complex compositions,microfabricated or micromachined particles, etc.

“Coupled” means bound. For example, a moiety can be coupled to amicroparticle by specific or nonspecific binding. As disclosed herein,the binding can be covalent or noncovalent, reversible or irreversible.

As used herein, “the moiety to be manipulated is substantially coupledonto surface of the binding partner” means that a percentage of themoiety to be manipulated is coupled onto a surface of the bindingpartner and can be manipulated by a suitable physical force viamanipulation of the binding partner. Ordinarily, at least 0.1% of themoiety to be manipulated is coupled onto a surface of the bindingpartner. Preferably, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80% or 90% of the moiety to be manipulated is coupled onto a surface ofthe binding partner.

As used herein, “the moiety to be manipulated is completely coupled ontosurface of the binding partner” means that at least 90% of the moiety tobe manipulated is coupled onto surface of the binding partner.Preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%of the moiety to be manipulated is coupled onto a surface of the bindingpartner.

A “specific binding member” is one of two different molecules having anarea on the surface or in a cavity which specifically binds to and isthereby defined as complementary with a particular spatial and chemicalorganization of the other molecule. A specific binding member can be amember of an immunological pair such as antigen-antibody orantibody-antibody, can be biotin-avidin, biotin-streptavidin, orbiotin-neutravidin, ligand-receptor, nucleic acid duplexes, IgG-proteinA, DNA-DNA, DNA-RNA, RNA-RNA, and the like.

An “antibody” is an immunoglobulin molecule, and can be, as nonlimitingexamples, an IgG, an IgM, or other type of immunoglobulin molecule. Asused herein, “antibody” also refers to a portion of an antibody moleculethat retains the binding specificity of the antibody from which it isderived (for example, single chain antibodies or Fab fragments).

A “nucleic acid molecule” is a polynucleotide. A nucleic acid moleculecan be DNA, RNA, or a combination of both. A nucleic acid molecule canalso include sugars other than ribose and deoxyribose incorporated intothe backbone, and thus can be other than DNA or RNA. A nucleic acid cancomprise nucleobases that are naturally occurring or that do not occurin nature, such as xanthine, derivatives of nucleobases, such as2-aminoadenine, and the like. A nucleic acid molecule of the presentinvention can have linkages other than phosphodiester linkages. Anucleic acid molecule of the present invention can be a peptide nucleicacid molecule, in which nucleobases are linked to a peptide backbone. Anucleic acid molecule can be of any length, and can be single-stranded,double-stranded, or triple-stranded, or any combination thereof.

“Homogeneous manipulation” refers to the manipulation of particles in amixture using physical forces, wherein all particles of the mixture havethe same response to the applied force.

“Selective manipulation” refers to the manipulation of particles usingphysical forces, in which different particles in a mixture havedifferent responses to the applied force, including situations where aforce used for manipulation of one particle has no effect on otherparticles.

A “fluid sample” is any fluid from which components are to be separatedor analyzed. A sample can be from any source, such as an organism, groupof organisms from the same or different species, from the environment,such as from a body of water or from the soil, or from a food source oran industrial source. A sample can be an unprocessed or a processedsample. A sample can be a gas, a liquid, or a semi-solid, and can be asolution or a suspension. A sample can be an extract, for example aliquid extract of a soil or food sample, an extract of a throat orgenital swab, or an extract of a fecal sample, or a wash of an internalarea of the body.

A “blood sample” as used herein can refer to a processed or unprocessedblood sample, i.e., it can be a centrifuged, filtered, extracted, orotherwise treated blood sample, including a blood sample to which one ormore reagents such as, but not limited to, anticoagulants or stabilizershave been added. An example of blood sample is a buffy coat that isobtained by processing human blood for enriching white blood cells.Another example of a blood sample is a blood sample that has been“washed” to remove serum components by centrifuging the sample to pelletcells, removing the serum supernatant, and resuspending the cells in asolution or buffer. Other blood samples include cord blood samples, bonemarrow aspirates, internal blood or peripheral blood. A blood sample canbe of any volume, and can be from any subject such as an animal orhuman. A preferred subject is a human.

A “rare cell” is a cell that is either 1) of a cell type that is lessthan 1% of the total nucleated cell population in a fluid sample, or 2)of a cell type that is present at less than one million cells permilliliter of fluid sample. A “rare cell of interest” is a cell whoseenrichment is desirable.

A ‘desired cell’ or ‘target cell’ is a specific type of rare cell ofinterest that might be identified and/or separated and/or enriched fromother types of cells by methods involving a marker or property that ispreferentially present or absent on the desired or target cells. Cellsthat are desirably removed in order to facilitate the identification,isolation, or characterization of a target cell are referred to as“non-target cells”, and a biological sample may contain multiple typesof non-target cells; accordingly, the methods of the invention can beused in various combinations to remove different non-target cells. Forexample, more than one type of cell may be removed by use of one or moreselective lysis steps; more than one type of non-target cell may beremoved by selective binding to a solid support, and this may involveindividual steps, each using a specific binding member to remove onecell type, or it may involve a single processing step where the sampleis exposed to more than one specific binding member adhered to one ormore solid surfaces, to effect removal of more than one type ofnon-target cell in one step.

“Without removing a target cell” as used herein means that the specificstep or combination of steps removes no more than 50% of the target cellpopulation from a sample, when the target cell type is present. This canbe determined by spiking a sample with a known number of target cells todetermine how many are recovered for detection; as a standard, theefficiency of recovery can be determined by spiking a sample with 5-10target cells and determining how many of those cells are detected afterthe enrichment process. Preferably the step or combination of stepsremoves less than 50% of the target cells from the sample, and typicallythe present methods remove less than 35% of the target cells. In someembodiments, the combination of steps used to isolate a target cellpopulation from a sample remove on average less than about 30% of thetarget cells, or less than 20% of the target cells. In some embodiments,the steps or the overall method remove less than 10% of the targetcells, or less than 5% of the target cells, or less than about 1% of thetarget cells.

As used herein, “removing a non-target cell” or cell type means that atleast a majority of the non-target cell population is removed by thestep. Cells are removed when they are taken out of the sample, or whenthey are lysed so that they would no longer be recognized as cells andare readily separated from the target cells by well known methods suchas filtration or centrifugation, which separate intact cells fromsmaller debris. The individual steps for removing a non-target cell typecan be repeated to further remove the non-target cell and further enrichthe target cells in the sample.

A “white blood cell” or “WBC” is a leukocyte, or a cell of thehematopoietic lineage that is not a reticulocyte or platelet and thatcan be found in the blood of an animal or human. Leukocytes can includenatural killer cells (“NK cells”) and lymphocytes, such as B lymphocytes(“B cells”) or T lymphocytes (“T cells”). Leukocytes can also includephagocytic cells, such as monocytes, macrophages, and granulocytes,including basophils, eosinophils and neutrophils. Leukocytes can alsocomprise mast cells.

A “red blood cell” or “RBC” is an erythrocyte. Unless designated a“nucleated red blood cell” (“nRBC”) or “fetal nucleated red blood cell”or nucleated fetal red blood cell, as used herein, “red blood cell” isused to mean a non-nucleated red blood cell.

“Neoplastic cells” or “tumor cells” or “cancer cells” refers to abnormalwas are or were part of a tumor, or the progeny of such cells. Thesecells tend to show partial or complete lack of structural organizationand functional coordination with the normal tissue, and may be benign ormalignant. Cancer cells, unlike benign tumor cells, exhibit theproperties of invasion and metastasis and are highly anaplastic. Cancercells include the two broad categories of carcinoma and sarcoma.

A “tumor” or “neoplasm” is an abnormal growth of tissue resulting fromuncontrolled, progressive multiplication of cells and serving nophysiological function; a neoplasm

A “cancer” is any of various malignant neoplasms characterized by theproliferation of anaplastic cells that tend to invade surrounding tissueand metastasize to new body sites.

A “malignant cell” is a cell having the property of locally invasive anddestructive growth and distal metastasis. Examples of “malignant cells”include, but not limited to, leukemia cells, lymphoma cells, cancercells of solid tumors, metastatic solid tumor cells (e.g., breast cancercells, prostate cancer cells, lung cancer cells, colon cancer cells) invarious body fluids including blood, bone marrow, ascitic fluids, stool,urine, bronchial washes etc.

A “cancerous cell” is a cell that exhibits deregulated growth and, inmost cases, has lost at least one of its differentiated properties, suchas, but not limited to, characteristic morphology, non-migratorybehavior, cell-cell interaction and cell-signaling behavior, proteinexpression and secretion pattern, etc. In most cases, cancerous cellsare either of epithelial or of mesenchymal origin that aredistinguishable from normal epithelial cells.

“Cancer” refers to a neoplastic disease the natural course of which isfatal. Cancer cells, unlike benign tumor cells, exhibit the propertiesof invasion and metastasis and are highly anaplastic. Cancer cellsinclude the two broad categories of carcinoma and sarcoma.

A “stem cell” is an undifferentiated cell that can give rise, throughone or more cell division cycles, to at least one differentiated celltype.

A “progenitor cell” is a committed but undifferentiated cell that cangive rise, through one or more cell division cycles, to at least onedifferentiated cell type. Typically, a stem cell gives rise to aprogenitor cell through one or more cell divisions in response to aparticular stimulus or set of stimuli, and a progenitor gives rise toone or more differentiated cell types in response to a particularstimulus or set of stimuli.

An “etiological agent” refers to any etiological agent, such as abacteria, fungus, protozoan, virus, parasite or prion that can infect asubject. An etiological agent can cause symptoms or a disease state inthe subject it infects. A human etiological agent is an etiologicalagent that can infect a human subject. Such human etiological agents maybe specific for humans, such as a specific human etiological agent, ormay infect a variety of species, such as a promiscuous human etiologicalagent.

“Subject” refers to any organism, such as an animal or a human. Ananimal can include any animal, such as a feral animal, a companionanimal such as a dog or cat, an agricultural animal such as a pig or acow, or a pleasure animal such as a horse.

A “chamber” is a structure that is capable of containing a fluid sample,in which at least one processing step can be performed. The chamber mayhave various dimensions and its volume may vary between ten microlitersand 0.5 liter.

A “filtration chamber” is a chamber through which or in which a fluidsample can be filtered.

A “filter” is a structure that comprises one or more pores or slots ofparticular dimensions (that can be within a particular range), thatallows the passage of some sample components but not others from oneside of the filter to the other, based on the size, shape, and/ordeformability of the particles. A filter can be made of any suitablematerial that prevents passage of insoluble particles, such as metal,ceramics, glass, silicon, plastics, polymers, fibers (such as paper orfabric), etc.

A “filtration unit” is a filtration chamber and the associated inlets,valves, and conduits that allow sample and solutions to be introducedinto the filtration chamber and sample components to be removed from thefiltration chamber. A filtration unit optionally also comprises aloading reservoir.

A “cartridge” is a structure that comprises at least one chamber that ispart of a manual or automated system and one or more conduits for thetransport of fluid into or out of at least one chamber. A cartridge mayor may not comprise one or more chips.

An “automated system for separating rare cells from a fluid sample” oran “automated system” is a device that comprises at least one filtrationchamber, automated means for directing fluid flow through the filtrationchamber, and at least one power source for providing fluid flow and,optionally, means for providing a signal source for the generation offorces on active chips. An automated system of the present invention canalso optionally include one or more active chips, separation chambers,separation columns, or permanent magnets.

A “port” is an opening in the housing of a chamber through which a fluidsample can enter or exit the chamber. A port can be of any dimensions,but preferably is of a shape and size that allows a sample to bedispensed into a chamber by pumping a fluid through a conduit, or bymeans of a pipette, syringe, or other means of dispensing ortransporting a sample.

An “inlet” is a point of entrance for sample, solutions, buffers, orreagents into a fluidic chamber. An inlet can be a port of a chamber, orcan be an opening in a conduit that leads, directly or indirectly, to achamber of an automated system.

An “outlet” is the opening at which sample, sample components, orreagents exit a fluidic chamber. The sample components and reagents thatleave a chamber can be waste, i.e., sample components that are not to beused further, or can be sample components or reagents to be recovered,such as, for example, reusable reagents or target cells to be furtheranalyzed or manipulated. An outlet can be a port of a chamber, butpreferably is an opening in a conduit that, directly or indirectly,leads from a chamber of an automated system.

A “conduit” is a means for fluid to be transported from a container to achamber of the present invention. Preferably a conduit directly orindirectly engages a port in the housing of a chamber. A conduit cancomprise any material that permits the passage of a fluid through it.Conduits can comprise tubing, such as, for example, rubber, Teflon, ortygon tubing. Conduits can also be molded out of a polymer or plastic,or drilled, etched, or machined into a metal, glass or ceramicsubstrate. Conduits can thus be integral to structures such as, forexample, a cartridge of the present invention. A conduit can be of anydimensions, but preferably ranges from 10 microns to 5 millimeters ininternal diameter. A conduit is preferably enclosed (other than fluidentry and exit points), or can be open at its upper surface, as acanal-type conduit.

“Enrich” means increase the relative concentration of a sample componentof a sample relative to other sample components (which can be the resultof reducing the concentration of other sample components), or increasethe absolute concentration of a sample component. For example, as usedherein, “enriching” nucleated fetal cells from a blood sample includesincreasing the proportion of nucleated fetal cells to all cells in theblood sample, enriching cancer cells of a blood sample can meanincreasing the concentration of cancer cells in the sample (for example,by reducing the sample volume) or reducing the concentration or numberof other cellular components of the blood sample to increase thepercentage of cells present that are cancer cells, and “enriching”cancer cells in a urine sample can mean increasing their concentrationin the sample such as by reducing sample volume or reducing the numberof ‘non-cancer’ cells in the sample.

“Separation” is a process in which one or more components of a sampleare spatially separated from one or more other components of a sample. Aseparation can be performed such that one or more sample components ofinterest is translocated to or retained in one or more areas of aseparation apparatus and at least some of the remaining components aretranslocated away from the area or areas where the one or more samplecomponents of interest are translocated to and/or retained in, or inwhich one or more sample components is retained in one or more areas andat least some or the remaining components are removed from the area orareas. Alternatively, one or more components of a sample can betranslocated to and/or retained in one or more areas and one or moresample components can be removed from the area or areas. It is alsopossible to cause one or more sample components to be translocated toone or more areas and one or more sample components of interest or oneor more components of a sample to be translocated to one or more otherareas. Separations can be achieved through, for example, filtration, orthe use of physical, chemical, electrical, or magnetic forces.Nonlimiting examples of forces that can be used in separations aregravity, mass flow, dielectrophoretic forces, traveling-wavedielectrophoretic forces, and electromagnetic forces.

“Separating a sample component from a (fluid) sample” means separating asample component from other components of the original sample, or fromcomponents of the sample that are remaining after one or more processingsteps. “Removing a sample component from a (fluid) sample” meansremoving a sample component from other components of the originalsample, or from components of the sample that are remaining after one ormore processing steps.

“Capture” is a type of separation in which one or more moieties orsample components is retained in or on one or more areas of a surface,chamber, chip, bead particles, tube, or any vessel that contains asample, where the remainder of the sample can be removed from that area.

An “assay” is a test performed on a sample or a component of a sample.An assay can test for the presence of a component, the amount orconcentration of a component, the composition of a component, theactivity of a component, etc. Assays that can be performed inconjunction with the compositions and methods of the present inventioninclude, but not limited to, immunocytochemical assays, interphase FISH(fluorescence in situ hybridization), karyotyping, immunological assays,biochemical assays, binding assays, cellular assays, genetic assays,gene expression assays and protein expression profiling assays.

A “binding assay” is an assay that tests for the presence orconcentration of an entity by detecting binding of the entity to aspecific binding member, or that tests the ability of an entity to bindanother entity, or tests the binding affinity of one entity for anotherentity. An entity can be an organic or inorganic molecule, a molecularcomplex that comprises, organic, inorganic, or a combination of organicand inorganic compounds, an organelle, a virus, or a cell. Bindingassays can use detectable labels or signal generating systems that giverise to detectable signals in the presence of the bound entity. Standardbinding assays include those that rely on nucleic acid hybridization todetect specific nucleic acid sequences, those that rely on antibodybinding to entities, and those that rely on ligands binding toreceptors.

A “biochemical assay” is an assay that tests for the presence,concentration, or activity of one or more components of a sample.

A “cellular assay” is an assay that tests for a cellular process, suchas, but not limited to, a metabolic activity, a catabolic activity, anion channel activity, an intracellular signaling activity, areceptor-linked signaling activity, a transcriptional activity, atranslational activity, or a secretory activity.

A “genetic assay” is an assay that tests for the presence or sequence ofa genetic element, where a genetic element can be any segment of a DNAor RNA molecule, including, but not limited to, a gene, a repetitiveelement, a transposable element, a regulatory element, a telomere, acentromere, or DNA or RNA of unknown function. As nonlimiting examples,genetic assays can be gene expression assays, PCR assays, karyotyping,or FISH. Genetic assays can use nucleic acid hybridization techniques,can comprise nucleic acid sequencing reactions, or can use one or moreenzymes such as polymerases, as, for example a genetic assay based onPCR. A genetic assay can use one or more detectable labels, such as, butnot limited to, fluorochromes, radioisotopes, or signal generatingsystems.

“Immunostaining” refers to staining of a specific antigen or structuresuch as a cell by any method in which the stain (or stain-generatingsystem, or signal-generating system) is complexed with a specificantibody.

“Polymerase chain reaction” or “PCR” refers to method for amplifyingspecific sequences of nucleotides (amplicon). PCR depends on the abilityof a nucleic acid polymerase, preferably a thermostable one, to extend aprimer on a template containing the amplicon. RT-PCR is a PCR based on atemplate (cDNA) generated from reverse transcription from mRNA preparedfrom a sample. Quantitative Reverse Transcription PCR (qRT-PCR) or theReal-Time RT-PCR is a RT-PCR in which the RT-PCR products for eachsample in every cycle are quantified.

“FISH” or “fluorescence in situ hybridization” is an assay wherein agenetic marker can be localized to a chromosome by hybridization.Typically, to perform FISH, a nucleic acid probe that is fluorescentlylabeled is hybridized to interphase chromosomes that are prepared on aslide. The presence and location of a hybridizing probe can bevisualized by fluorescence microscopy. The probe can also include anenzyme and be used in conjunction with a fluorescent enzyme substrate.

“Karyotyping” refers to the analysis of chromosomes that includes thepresence and number of chromosomes of each type (for example, each ofthe 24 chromosomes of the human haplotype (chromosomes 1-22, X, and Y)),and the presence of morphological abnormalities in the chromosomes, suchas, for example, translocations or deletions. Karyotyping typicallyinvolves performing a chromosome spread of a cell in metaphase. Thechromosomes can then be visualized using, foe example, but not limitedto, stains or genetic probes to distinguish the specific chromosomes.

A “gene expression assay” (or “gene expression profiling assay”) is anassay that tests for the presence or quantity of one or more geneexpression products, i.e. messenger RNAs. The one or more types of mRNAscan be assayed simultaneously on cells of the interest from a sample.For different applications, the number and/or the types of mRNAmolecules to be assayed in the gene expression assays may be different.

A “protein expression assay” (or “protein expression profiling assay”)is an assay that tests for the presence or quantity of one or moreproteins. One or more types of protein can be assayed simultaneously onthe cells of the interest from a sample. For different applications, thenumber and/or the types of protein molecules to be assayed in theprotein expression assays may be different.

“Histological examination” refers to the examination of cells usinghistochemical or stains or specific binding members (generally coupledto detectable labels) that can determine the type of cell, theexpression of particular markers by the cell, or can reveal structuralfeatures of the cell (such as the nucleus, cytoskeleton, etc.) or thestate or function of a cell. In general, cells can be prepared on slidesand “stained” using dyes or specific binding members directly orindirectly bound to detectable labels, for histological examination.Examples of dyes that can be used in histological examination arenuclear stains, such as Hoescht stains, or cell viability stains, suchas Trypan blue, or cellular structure stains such as Wright or Giemsa,enzyme activity benzidine for HRP to form visible precipitate. Examplesof specific binding members that can be used in histological examinationof fetal red blood cells are antibodies that specifically recognizefetal or embryonic hemoglobin.

A “well” is a structure in a chip, with a lower surface surrounded on atleast two sides by one or more walls that extend from the lower surfaceof the well or channel. The walls can extend upward from the lowersurface of a well or channel at any angle or in any way. The walls canbe of an irregular conformation, that is, they may extend upward in asigmoidal or otherwise curved or multi-angled fashion. The lower surfaceof the well or channel can be at the same level as the upper surface ofa chip or higher than the upper surface of a chip, or lower than theupper surface of a chip, such that the well is a depression in thesurface of a chip. The sides or walls of a well or channel can comprisematerials other than those that make up the lower surface of a chip.

A “pore” is an opening in a surface, such as a filter of the presentinvention, that provides fluid communication between one side of thesurface and the other. A pore can be of any size and of any shape, butpreferably a pore is of a size and shape that restricts passage of atleast one insoluble sample component from one side of a filter to theother side of a filter based on the size, shape, and deformability (orlack thereof), of the sample component.

“Continuous flow” means that fluid is pumped or injected into a chamberof the present invention continuously during the separation process.This allows for components of a sample that are not selectively retainedin a chamber to be flushed out of the chamber during the separationprocess.

“Binding partner” refers to any substances that both bind to themoieties with desired affinity or specificity and are manipulatable withthe desired physical force(s). Non-limiting examples of the bindingpartners include microparticles.

A “microparticle” is a structure of any shape and of any compositionthat is manipulatable by desired physical force(s). The microparticlesused in the methods could have a dimension from about 0.01 micron toabout ten centimeters. Preferably, the microparticles used in themethods have a dimension from about 0.1 micron to about several hundredmicrons. Such particles or microparticles can be comprised of anysuitable material, such as glass or ceramics, and/or one or morepolymers, such as, for example, nylon, polytetrafluoroethylene(TEFLON™), polystyrene, polyacrylamide, sepaharose, agarose, cellulose,cellulose derivatives, or dextran, and/or can comprise metals. Examplesof microparticles include, but are not limited to, magnetic beads,magnetic particles, plastic particles, ceramic particles, carbonparticles, polystyrene microbeads, glass beads, hollow glass spheres,metal particles, particles of complex compositions, microfabricatedfree-standing microstructures, etc. The examples of microfabricatedfree-standing microstructures may include those described in “Design ofasynchronous dielectric micromotors” by Hagedorn et al., in Journal ofElectrostatics, Volume: 33, Pages 159-185 (1994). Particles of complexcompositions refer to the particles that comprise or consists ofmultiple compositional elements, for example, a metallic sphere coveredwith a thin layer of non-conducting polymer film.

“A preparation of microparticles” is a composition that comprisesmicroparticles of one or more types and can optionally include at leastone other compound, molecule, structure, solution, reagent, particle, orchemical entity. For example, a preparation of microparticles can be asuspension of microparticles in a buffer, and can optionally includespecific binding members, enzymes, inert particles, surfactants,ligands, detergents, etc.

Other technical terms used herein have their ordinary meaning in the artthat they are used, as exemplified by a variety of technicaldictionaries.

The present invention recognizes that the diagnosis and monitoring ofcancer disease faces challenges posed by the multiplicity of locationsat which a cancer can develop in a patient and the fact that a smallnumber of cells can give rise to a tumor or metastasis. The shedding ofcancer cells into the bloodstream is a common characteristic of cancerdisease and is central to the present invention's ability to diagnoseand monitor cancer using biological fluid samples (such as bloodsamples) obtained from patients. Analysis of complex fluids, such asbiological fluid samples, can be confounded by many sample componentsthat can interfere with the analysis. Sample analysis can be even moreproblematic when the target of the analysis is a rare cell type, forexample, when the target cells are malignant cells present in the bloodof a patient. In processing such samples, it is often necessary to both“debulk” the sample, by reducing the volume to a manageable level, andto enrich the population of cancer cells that are the target ofanalysis. Procedures for the processing of fluid samples are often timeconsuming and inefficient. In some aspects, the present inventionprovides efficient methods for the enrichment of cancer cells from bloodsamples. In addition the present invention further recognizes that themolecular composition of a cancer cells can mutate or evolve during thecourse of the disease. Also the methods described in this invention aredesigned to very rapidly remove plasma proteins, most WBCs and RBCs withlesser undesired effects, resulting in easy detection of CTCs in anenriched sample. The current invention can be applied to separate plasmaprotein, enrich other rare cells, including stem cells, fetal cells,immune cells, etc, followed by downstream analysis, manipulations, andapplications such as flow cytometry, PCR, immunofluorescence,immunocytochemistry, image analysis, enzymatic assays, gene expressionprofiling analysis, efficacy tests of therapeutics, culturing ofenriched rare cells, and therapeutic use of enriched rare cells. Centralto the present invention are methods and compositions for the isolationand identification of cancer cells throughout the course of the diseaseand use of information about the presence and/or abundance of suchcancer cells in a method to detect, diagnose or prognose a cancer. Themethods of the present invention can overcome the variability andgenetic instability typical of cancer cells, thus providing a reliablediagnostics approach.

As a non-limiting introduction to the breadth of the present invention,the present invention includes several general and useful aspects,including:

1) a method for enriching a target cell, such as cancer cells, from abiological sample such as a blood sample. When applied to a bloodsample, the methods may comprise the selective removal of red bloodcells (RBCs) by lysis, and the selective removal of WBCs by binding themto a specific binding member and removing them, such as by precipitationor by allowing them to bind to a solid support via specific bindingmembers. Alternatively, the method may comprise the removal of whiteblood cells (WBCs) using microparticles to which a specific bindingmember that is specific for WBCs is affixed, and removal of red bloodcells (RBCs) from said blood sample to enrich rare cells with adensity-based approach e.g., a ficoll gradient centrifugation. Removalof WBCs and RBCs can be done in either order or both may be removed atthe same time.

2) methods and compositions for identifying and characterizing cancercells in an enriched cancer cell-containing sample obtained from a bloodsample, based on the molecular recognition and labeling of selectedcancer markers.

3) a method for diagnosing and monitoring cancer disease or itstreatment, comprising the measurement of the number or proportion orproperties of cancer cells in a blood sample obtained from a subject.

In certain embodiments, the target cell is a cancer cell or amesenchymal cell, and desirably the cancer or mesenchymal cell isdistinguished by the identification methods described herein from anynormal epithelial cells in the sample. Frequently, the sample is a bloodsample. Note that the methods involve enrichment of a rare cell typethat may or may not be present, and the presence, absence, number,proportion, or properties of any rare cells present are usuallydiagnostic; thus the method is useful regardless of whether or not rarecells are found in the sample. The methods are typically describedherein as though the target cell is present, but they are equally usefulto detect the absence of a target cell. While the target cell isolationand identification methods described herein are highly specific for thetarget cells of interest, other cells may also be identified in somesamples as target-like; such methods are nevertheless useful fordiagnostic and other purposes provided that information about the numberor fraction of non-target cells being identified can be determined, asby comparison to samples from subjects having no target cells present.In some embodiments, further characterization methods may be used toreduce the number of target-like cells that are detected along with thetarget cells.

In some embodiments, the enrichment of the target cell(s) in the sampleis achieved in a series of steps. One step in the sequence may be aselective lysis step, which uses osmotic pressure change to selectivelylyse certain types of cells, such as red blood cells, without lysing thetarget cell. That enables the removal of at least one type of non-targetcell, so that the remaining cell population in the sample is enriched inthe target cell.

Typically, the enrichment of the target cell(s) in the sample involvesselective removal of at least one non-target cell type by adhering cellsof the non-target type to a solid surface. This can be accomplished byaffixing a selective binding member to the solid surface, whichselective binding member has a high affinity for a non-target cell typeto be removed and has little or no effective affinity for the targetcell type. The sample is then exposed to the solid surface having anaffixed selective binding member, and at least one non-target cell typeis thus removed from the sample, which is thereby enriched in the targetcell(s), if any are present.

Frequently, the selective binding member is an antibody or antibodyfragment that is selective for a cell surface marker that is associatedwith a non-target cell and is expected to be absent on the target cells.An example of such markers would be a surface antigen that occurs on oneor more types of hematopoietic cells, when the target cell is anon-hematopoietic cell. The solid surface can be a surface of acontainer that the sample is placed in, or a surface of a swab or otheritem to be passed through the sample, or it can be a finely dividedmaterial such as a bead or gel that can be admixed with the sample. Ineach case, the sample is exposed to the solid surface for a sufficientperiod of time to permit binding of the non-target cell type to beremoved to the specific binding member; then the sample is separatedfrom the solid surface for further enrichment or analysis. In onepreferred embodiment, the solid surface is a plurality of beads, such asmagnetic beads, that provide a high surface area for affixing thespecific binding member of interest, can be efficiently mixed with thesample to permit binding of the non-target cell to the specific bindingmaterial, and can be conveniently separated from the sample to removethe non-target cells.

These aspects of the invention, as well as others described herein, canbe achieved by using the methods, articles of manufacture andcompositions of matter described herein. It will be further recognizedthat various aspects of the present invention can be combined to makedesirable embodiments of the invention. To gain a full appreciation ofthe scope of the present invention, certain non-limiting examples aredescribed herein.

I. Method of Enriching Cancer Cells of a Fluid Sample Using Depletion ofRed Blood Cells and White Blood Cells.

One aspect of the present invention includes methods and compositionsfor enriching rare cells or target cells by removal of plasma proteins,RBCs and WBCs from a biological sample by means of combining densitybased centrifugation and immuno-particle methods to remove non-targetcells and to enrich rare cells such as circulating tumor cells, stemcells fetal cells, immune cells, and other rare cells with the followingexemplary steps: a) removing WBCs with anti-body coated immunoparticles:b) removing RBCs with a density-based approach, and c) performance asubsequent analysis, manipulation or application of the rare cell ortarget cell. The density-based approach can also be used to remove othermaterials such as non-cellular debris and proteins from the targetcells.

In one specific embodiment, the present method can be used for depletionof plasma proteins, RBCs and WBCs by means of combining density basedcentrifugation and immunoparticles to enrich rare cells includingcirculating tumor cells, stem cells, fetal cells, immune cells, andother rare cells from a blood sample.

In some embodiments, the blood sample is collected in any suitableanti-coagulant containing tube, which may optionally also include EDTA,ACD, Heparin, Cyto-chex and similar known materials for stabilizing orpreserving the blood sample.

In some embodiments, a density-based centrifugation method is used toremove at least one type of non-target cell. The medium for such densitybased centrifugation separation could have a range of suitableconcentrations to generate same density and/or continuous ordiscontinuous gradient, polysucrose based reagent such as Ficoll, or anyother reagents applied to prepare desired density to separate proteinsand or cells by centrifugation.

In a certain embodiment of the present invention, the immuno-particlescould be any solid phase particle or microspheres such as magneticparticle, sepharose, sephadex, and agarose etc based particles, and theparticles can be chemically modified to conjugate to an antibody or anyother specific binding members for selection and binding of non-targetcell types to be removed as part of the process for enrichment of thesample in the target cell type of interest.

The enriched cells, and/or separated plasma proteins, and/or white bloodcell population are available for various analysis and applications,including immunocytometry, flow cytometry, PCR (e.g., ReverseTranscription-PCR and the real time Reverse Transcription-PCR), imageanalysis, immunofluorescence, genotyping, gene profiling examination,culturing of enriched cells, mass-spectrometry and other cell and/orprotein related studies, cell-based therapy, cell-based assays,enzymatic assays, etc.

One exemplary method for enriching tumor cells from a blood sample isoutlined below:

A blood sample is diluted with an equal volume of phosphate bufferedsaline (PBS) or Hank's buffered saline solution, or any buffer withosmolarity of 270-330 milliosmoles per kilogram (mOsm/kg), or mediumsuch as cell culture medium. 0.1-0.5 ml of antibody-coatedimmunoparticles is added in diluted blood, followed by gentle shaking atroom temperature for 5 minutes or longer. This mixture is subsequentlyloaded on the top of a separation medium (e.g., Ficoll diluted in arange between 80-98% using above mentioned buffer or medium), followedby centrifugation between 200-800 g for 5 minutes or longer at roomtemperature. Plasma, white blood cells, and Ficoll at different layersare collected from the top. Either a white blood cell layer or alllayers pooled together are centrifuged at 800-1500 g for 5 min orlonger. A cell pellet that will contain the target cells if any werepresent in the sample is prpareds following this procedure and isresuspended and subjected to subsequent analysis.

In one embodiment, the present invention is directed to a method fordetecting a non-hematopoietic cancer cell, in a blood sample, whichmethod comprises: a) providing a blood sample; b) removing red bloodcells (RBCS) from said blood sample, with a density-based approach,e.g., a Ficoll density centrifugation, and removing white blood cells(WBCs) from said blood sample to enrich a non-hematopoietic cell type,e.g., a non-hematopoietic tumor cell, if any, from said blood sample,with a microparticle-based approach; and c) assessing the presence,absence and/or amount of said enriched non-hematopoietic cell or tumorcell. It is apparent to those skilled in the art that this method isequally useful where the non-target cell happens to be absent from asample, because the recovery of target cells is sufficiently efficientthat the absence of detected target cells is a diagnostically meaningfuland useful result, just as the presence or number of target cells isdiagnostically useful.

One of the possible alternative methods is achieved by the followingsteps:

A blood sample is diluted with an equal volume of phosphate bufferedsaline or Hank's buffered saline solution, or any buffer with osmolarityof 270-330 mOsm/kg, or a medium such as cell culture medium, andsubsequently loaded on the top of a density-based separation medium(e.g., Ficoll diluted in a range between 80-98% using above mentionedbuffer or medium), followed by centrifugation between 200-800 g for 5minutes or longer at a convenient temperature such as room temperature.Plasma, white blood cell, and Ficoll at different layers are collectedfrom the top. 0.1-0.5 ml of antibody-coated immunoparticles are added ineither white blood cell layer or pooled all layers, followed by gentleshaking for 5 minutes or longer at room temperature. Immunoparticleswith bound WBCs are separated by either magnet or centrifugation at200-800 g for 5 minutes or longer. Collected supernatants arecentrifuged again at 800-1500 g for 5 min or longer. The cell pelletfrom this procedure is resuspended and subjected to subsequent analysis.

The present invention also provides methods and compositions fordetecting a non-hematopoietic cell, e.g., a non-hematopoietic cancercell, in a blood sample. In one embodiment, the present invention isdirected to a method for detecting a non-hematopoietic cancer cell, in ablood sample, which method comprises: a) providing a blood sample; b)removing red blood cells (RBCs) from said blood sample, with the provisothat said RBCs are not removed from said blood sample via acentrifugation, e.g., a Ficoll gradient centrifugation, and removingwhite blood cells (WBCs) from said blood sample to enrich anon-hematopoietic cell, e.g., a non-hematopoietic tumor cell, if any,from said blood sample; and c) assessing the presence, absence and/oramount of said enriched non-hematopoietic cell or tumor cell.

The present method can be used to detect a cancer cell in any suitablesample, and is frequently used to detect circulating tumor or cancercells in a blood sample or other clinical sample. For example, a sampleto be tested can be a whole blood sample or a peripheral blood sample.

The present method can be used to detect any suitable non-hematopoieticcell, e.g., a non-hematopoietic cancer cell, in a blood sample. Forexample, the non-hematopoietic cell to be detected can be a cancerouscell or a cancer cell. In some embodiments, the method is used tospecifically identify cancerous or mesenchymal cells, and to distinguishthem from any normal epithelial cells that may be present.

The RBCs can be removed from a blood sample by any suitable methods. Forexample, the RBCs can be removed by sedimentation, filtration, selectivelysis, or binding to a specific binding member that specifically bindsRBCs a combination of the above, and/or repetition of the above. In somepreferred embodiments, RBCs are removed from a blood sample by lysingthem by conventional methods, such as exposing them to a medium orbuffer known to selectively lyse RBCs without lysing the target cells ofinterest.

The WBCs can be removed by any suitable methods. For example, the WBCscan be removed by binding to a specific binding member that specificallybinds WBCs, with significantly reduced or no binding to the targetcells. Any suitable specific binding members can be used. In onespecific embodiment, the specific binding member can be an antibody thatspecifically binds to a component on the surface of WBCs. Such exemplaryantibodies include an antibody that specifically binds to CD3, CD11b,CD14, CD17, CD31, CD34, CD45, CD50, CD53, CD63, CD69, CD81, CD84, CD102or CD166. In some embodiments, the antibody is one that specificallybinds to CD50.

The specific binding member that is used to remove WBCs can be used in asolution or can be bound to a solid surface such as a particle. Thespecific binding member can be bound directly or indirectly to a solidsurface. For example, the specific binding member can be boundindirectly to a solid surface through another binding pair, e.g., abiotin-avidin/strepavidin binding pair. The specific binding member canbe bound to a solid surface with or without any prior chemicalmodification(s) and/or conjugation to any molecules such as a member ofseparate binding pair. Any suitable solid surface can be used. Forexample, a specific binding member can be bound to a magnetic particleand the RBCs and/or WBCs bound to the magnetic particle can be removedfrom a blood sample using a magnetic field or force.

The RBCs and WBCs can be removed from a blood sample in any suitableorder. For example, the RBCs can be removed before the WBCs are removedfrom the blood sample. In another example, the WBCs are removed beforethe RBCs are removed from the blood sample. In still another example,the RBCs and WBCs can be removed from a blood sample simultaneously.

To further enrich the tumor cells to be assessed, the present methodscan further comprise removing a component(s) other than the RBCs andWBCs. For example, the present methods can comprise removing platelets,stem cells, stromal cells, endothelial cells or soluble proteins fromthe blood sample. The removal of the RBCs and WBCs and the removal ofthe other undesirable component(s) can be carried out in separate stepsor in the same step. Methods for such removal could include thosedescribed below as well as others known to those skilled in the art.

A suitable method for enriching tumor cells from a blood sample isoutlined below:

A sample of 10 mL of blood, stored at room temperature for up to 7 days,preferably up to 3 days, is transferred to a 50 mL centrifugation tubeand the volume is adjusted to 30 mL with a solution consisting of: 5 mMEDTA, 1% BSA in Hanks Balanced Salt Solution (HBSS), and PBS The sampleis centrifuged at 1400 rpm for 5 min at room temperature. 23 mL ofsupernatant are discarded and the pellet is completely resuspended bygentle shaking. An RBC lysis buffer (one liter of 10× stock for thisbuffer can be made with NH₄C_(1-82.9) g (Sigma, Cat # A-0171), KHCO3—10g (Sigma, Cat #237205-500 g), EDTA—2 ml of 0.5M EDTA (Molecular Probes,Cat #15575-020), pH 7.2, vacuum filtered) is added (22 mL), and thesample is incubated with rotation at room temperature for about 8 min.Following the lysis of the red blood cells the sample is centrifuged at1400 rpm for 5 min at room temperature. The supernatant is aspirated,leaving remaining cell pellet undisturbed. The cell pellet is completelyresuspended in 45 mL of solution containing 5 mM EDTA, 1% BSA in HBSS,and PBS. The sample is centrifuged again at 1400 rpm for 5 min at roomtemperature; the supernatant is discarded and the cells are resuspendedin 0.3 mL of solution containing 5 mM EDTA, 1% BSA in HBSS, and PBS. Thecell suspension is transferred into a 2 ml (U-bottom) eppendorf tube,and 0.8 mL of magnetic beads slurry is added. The beads are coated withantibody recognizing the CD50 antigen. The cells/beads suspension isincubated with gentle rotation for 5 to 60 minutes at room temperature.Following the incubation, the tube is positioned on a magnetic stand atroom temperature for 1 min with lead cap open, to allow for the beadsand the non-cancer cells adsorbed to the beads to migrate towards theside of the tube facing the magnet. The solution is carefullytransferred to a 1.7 mL eppendorf tube (V-bottom). The enriched samplecontaining the tumor cells is centrifuged at 10,000 rpm for 1 minute.After discarding the supernatant, the cells are resuspendend in 40 μL ofPBS buffer.

In a variant of the method described above, the removal of thenon-cancer cells is achieved by the following method:

Following the lysis of the red blood cells the sample is centrifuged at1400 rpm for 5 min at room temperature. The supernatant is aspirated,leaving a remaining cell pellet. The cell pellet is completelyresuspended in 45 mL of solution containing 5 mM EDTA, 1% BSA in HBSS,PBS. The sample is centrifuged again at 1400 rpm for 5 min at roomtemperature; the supernatant is discarded and the cells are resuspendedin 0.3 mL of solution containing 5 mM EDTA, 1% BSA in HBSS, and PBS. Thecell suspension is transferred into a 2 ml (U-bottom) eppendorf tube and0.8 mL of magnetic beads slurry is added. The beads are coated withantibodies recognizing LD50, and optionally CD34 and CD31 and CD235aantigens. The cells/beads suspension is incubated with gentle rotationfor 5 to 60 minutes at room temperature. Following the incubation, thetube is positioned on a magnetic stand at room temperature for 1 minwith lead cap open, to allow for the beads and the hematopoietic cellsadsorbed to the beads to migrate towards the side of the tube facing themagnet. The solution is carefully transferred to a 1.7 mL eppendorf tube(V-bottom). The enriched sample is centrifuged at 10,000 rpm for 1minute. After discarding the supernatant, the cells are re-suspendend in40 μL of PBS buffer.

Other antibodies that specifically bind to non-target cells likely to bepresent in the sample can, of course, also be affixed to a surface orbead and similarly used to remove non-target cells and further enrichthe sample.

To further enrich the tumor cells to be assessed, the present methodscan comprise debulking the blood sample. Any suitable debulking methodcan be used. For example, the debulking step can comprise a filtrationstep, a centrifugation step or a selective sedimentation step. This stepmay be included before or after removal of RBCs, for example, and theprocess may include more than one such step as needed.

In one preferred embodiment of the present invention the enrichment ofthe non-hematopoietic cancer cells from a blood sample is performed byan automated instrument. Automated manipulation of biological samplesand fluids is most efficiently performed by avoiding centrifugation as ameans to debulk the samples, although centrifugation can also beintegrated into an automated process.

In one embodiment of the present invention the debulking of the samplesis performed using a “French press” device as described in FIG. 1. Thismethod is particularly appropriate for use in an automated system forprocessing multiple samples, as this debulking process is especiallysuited to automation. The “French press” device consists of a chamber,which in a preferred embodiment of the invention is of cylindrical shape(1 in FIG. 1), at least one filter (2 in FIG. 1), a piston (3 in FIG.1), and an inlet and outlet means (4 and 5 in FIG. 1) with valvescontrolling fluid flow. The filter contains at least one pore andpreferably contains a plurality of pores. The pores can be of any shapeand any suitable dimensions and cross-sectional shapes. For example, apore can be quadrilateral, rectangular, ellipsoid, or circular in shape,or of other geometric or non-geometric shape. A pore can have a diameter(or widest dimension) from about 0.1 micron to about 1000 microns,preferably from about 0.2 to about 5 microns, or about 1 to less than 10microns.

In a preferred embodiment, a pore is made during the machining of afilter, and is micro etched or bored into the filter material thatcomprises a hard, fluid-impermeable material such as glass, silicon,ceramics, metal or hard plastic such as acrylic, polycarbonate, orpolyimide. It is also possible to use a relatively non-hard surface forthe filter that is supported on a hard solid support. Another aspect ofthis invention is to modify the material by methods such as, but notlimited to, chemically or thermally modifying the material to siliconoxide, silicon nitride, plastics, or polymers. Preferably, however, thefilter comprises a hard material that is not substantially deformable bythe pressure used in generating fluid flow through the filter.

Preferably, the filter used for filtration in the present invention ismicrofabricated or micromachined filters so that the pores within afilter can achieve relatively precise and uniform dimensions. Suchprecise and uniform pore dimensions are a distinct advantage of themicrofabricated or micromachined filters of the present invention, incomparison with the conventional membrane filters made of materials suchas nylon, polycarbonate, polyester, mixed cellulose ester,polytetrafluoroethylene, polyethersulfone, etc. In the filters of thepresent invention, individual pores are isolated, have similar or almostidentical feature sizes, and are patterned on a filter. Such filtersallow precise separation of particles based on their sizes and otherproperties.

The filtration area of a filter is determined by the area of thesubstrate comprising the pores. The filtration area for microfabricatedfilters of the present invention can be between about 0.01 mm² and about0.1 m². Preferably, the filtration area is between about 0.25 mm² andabout 25 cm², and more preferably is between about 0.5 mm² and about 10cm². The variations of filtration areas allow the filters of theinvention to process sample volumes from about 100 microliters to about10 liters. The percent of the filtration area encompassed by pores canbe from about 1% to about 70%, preferably is from about 10% to about50%, and more preferably is from about 15 to about 40%. The filtrationarea of a microfabricated filter of the present invention can compriseany number of pores, and preferably comprises at least two pores, butmore preferably the number of pores in the filtration area of a filterof the present invention ranges from about 4 to about 1,000,000, andeven more preferably ranges from about 100 to about 250,000. Thethickness of the filter in the filtration area can range from about 10to about 1000 microns, but is preferably in the range of between about40 and about 500 microns.

The microfabricated filters of the present invention have pores that maybe etched through the filter substrate itself. The pores or openings ofthe filters can be made by using microfabrication or micromachiningtechniques on substrate materials, including, but not limited to,silicon, silicon dioxide, ceramics, glass, polymers such as polyimide,polyamide, etc. Various fabrication methods, as known to those skilledin the art of microlithography and microfabrication (See, for example,Rai-Choudhury P. (Editor), HANDBOOK OF MICROLITHOGRAPHY, MICROMACHININGAND MICROFABRICATION, Volume 2: Micromachining and microfabrication.SPIE Optical Engineering Press, Bellingham, Wash., USA (1997)), may beused. In many cases, standard microfabrication and micromachiningmethods and protocols may be involved. One example of suitablefabrication methods is photolithography involving single or multiplephotomasks. The protocols in the microfabrication may include many basicsteps, for example, photolithographic mask generation, deposition ofphotoresist, deposition of “sacrificial” material layers, photoresistpatterning with masks and developers, or “sacrificial” material layerpatterning. Pores can be made by etching into the substrate undercertain masking process so that the regions that have been masked arenot etched off and the regions that have not been mask-protected areetched off. The etching method can be dry-etching such as deep RIE(reactive ion etching), laser ablation, or can be wet etching involvingthe use of wet chemicals.

Preferably, appropriate microfabrication or micromachining techniquesare chosen to achieve a desired aspect ratio for the filter pores. Theaspect ratio refers to the ratio of the pore depth (corresponding to thethickness of the filter in the region of the pores) to the porediameter. The fabrication of filter pores with higher aspect ratios(i.e., greater pore depth) may involve deep etching methods. Manyfabrication methods, such as deep RIE, useful for the fabrication ofMEMS (micro electronic mechanical systems) devices can be used oremployed in making the microfabricated filters. The resulting pores can,as a result of the high aspect ratio and the etching method, have aslight tapering, such that their openings are narrower on one side ofthe filter than the other.

The present invention includes microfabricated filters comprising two ormore tapered pores. The substrate on which the filter pores, slots oropenings are fabricated or machined may be silicon, silicon dioxide,plastic, glass, ceramics or other solid materials. The solid materialsmay be porous or non-porous. Those who are skilled in microfabricationand micromachining fabrication may readily choose and determine thefabrication protocols and materials to be used for fabrication ofparticular filter geometries.

Using the microfabrication or micromachining methods, the filter slots,pores or openings can be made with precise geometries, and withsubstantially similar sizes. Depending on the fabrication methods ormaterials used, the accuracy of a single dimension of the filter slots(e.g. slot length, slot width) can be within 20%, or less than 10%, orless than 5%. Thus, the accuracy of the critical, single dimension ofthe filter pores (e.g. slot width for oblong or quadrilateral shapedslots) for the filters of the present invention are made with a sizevariation of, preferably, less than 2 microns, more preferably, lessthan 1 micron, or even more preferably less than 0.5 micron.

Preferably, filters of the present invention can be made using thetrack-etch technique, in which filters made of glass, silicon, silicondioxides, or polymers such as polycarbonate or polyester with discretepores having relatively-uniform pore sizes are made. For example, thefilter can be made by adapting and applying the track-etch techniquedescribed at whatman.com/products/nucleopore/tech frame.htm forNucleopore Track-etch membranes to filter substrates. In the techniqueused to make membrane filters, a thin polymer film is tracked withenergetic heavy ions to produce latent tracks on the film. The film isthen put in an etchant to produce pores, which etchant is frequently acaustic solution, but may be other etchants known in the art.

Preferred filters for the cell separation methods and systems of thepresent invention include microfabricated or micromachined filters thatcan be made with precise geometries for the openings on the filters.Individual openings are isolated with similar or almost identicalfeature sizes and are patterned on a filter. The openings can be ofdifferent shapes such as, for example, circular, quadrilateral, orelliptical. Such filters allow precise separation of particles based ontheir sizes and other properties.

In a preferred embodiment of a microfabricated filter, individual poresare isolated and of a cylindrical shape, i.e., they are substantiallycircular in one cross section parallel to the plane of the filter, andsubstantially rectangular in cross-section in the other direction,perpendicular to the filter, and the pore sizes in a filter aretypically within a 20% variation, where the pore size is calculated bythe smallest and largest dimension of the pore's cross-section (widthand length, respectively).

The present invention also includes methods of treating amicrofabricated filter to improve its filtering efficiency. In thesemethods, one or both surfaces of the filter is treated or coated ormodified to increase its filtering efficiency. In a preferred method,one or both surfaces of the filter is treated or modified to reduce thepossibility of sample components (such as but not limited to cells)interacting with or adhering to the filter.

A filter can be physically or chemically treated, for example, to alterits surface properties (e.g. hydrophobic, hydrophilic). For example, afilter can be heated or treated with oxygen plasma, modified to siliconnitride or can be treated with at least one acid or at least one base,to increase its hydrophilicity or surface charge. For example, a glassor silica filter can be heated to oxidize the surface of the filter.Heating times and temperatures can vary depending on the filter materialand the degree of oxidation desired. In one example, a glass filter canbe heated to a temperature of from about 200 to 1000 degrees Celsius forfrom about thirty minutes to twenty-four hours.

In another example, a filter can be treated with one or more acids orone or more bases to increase the hydrophilicity of the filter surface.In preferred embodiments, a filter that comprises glass or silica istreated with at least one acid.

An acid used in treating a filter of the present invention can be anyacid. As nonlimiting examples, the acid can be HCl, H₂SO₄, NaHSO₄, HSO₄,HNO₃, HF, H₃PO₄, HBr, HCOOH, or CH₃COOH. The acid can be of aconcentration about 0.1 N or greater, and preferably is about 0.5 N orhigher in concentration, and more preferably is greater than about 1 Nin concentration. For example, the concentration of acid preferably isfrom about 1 N to about 10 N. The incubation time can be from one minuteto days, but preferably is from about 5 minutes to about 2 hours.

Optimal concentrations and incubation times for treating amicrofabricated filter to increase its hydrophilicity can be determinedempirically. The microfabricated filter can be placed in a solution ofacid for any length of time, preferably for more than one minute, andmore preferably for more than about five minutes. Acid treatment can bedone under any non-freezing and non-boiling temperature, preferably at atemperature greater than or equal to room temperature.

Alternatively or in addition, a microfabricated filter of the presentinvention can be treated with a base, such as a basic solution, that cancomprise, as nonlimiting examples, NaOH, KOH, Ba(OH)₂, LiOH, CsOH, orCa(OH)₂. The basic solution can be of a concentration of about 0.01 N orgreater, and preferably is greater than about 0.05 N, and morepreferably greater than about 0.1 N in concentration. The ion transportmeasuring means can be placed in a solution of base for any length oftime, preferably for more than one minute, and more preferably for morethan about five minutes. Base treatment can be done under any non-frozenand non-boiling temperature, preferably at a temperature greater than orequal to room temperature.

The effectiveness of a physical or chemical treatment in increasing thehydrophilicity of a filter surface can be tested by measuring the spreadof a drop of water placed on the surface of a treated and non-treatedfilter, where increased spreading of a drop of uniform volume indicatesincreased hydrophilicity of a surface. The effectiveness of a filtertreatment can also be tested by incubating a treated filter with cellsor biological samples to determine the degree of sample componentadhesion to the treated filter.

In another embodiment, the surface of a filter, such as but not limitedto a polymeric filter, can be chemically treated to alter the surfaceproperties of the filter. For example, the surface of a glass, silica,or polymeric filter can be derivatized by any of various chemicaltreatments to add chemical groups that can decrease the interaction ofsample components with the filter surface

One or more compounds can also be adsorbed onto or conjugated to thesurface of a microfabricated filter made of any suitable material, suchas, for example, one or more metals, one or more ceramics, one or morepolymers, glass, silica, silicon dioxide, or combinations thereof. Inpreferred embodiments of the present invention, the surface or surfacesof a microfabricated filter of the present invention is coated with acompound to increase the efficiency of filtration by reducing theinteraction of sample components with the filter surface.

For example, the surface of a filter can be coated with a molecule, suchas, but not limited to, a protein, peptide, or polymer, includingnaturally occurring or synthetic polymers. The material used to coat thefilter is preferably biocompatible, meaning it does not have deleteriouseffects on cells or other components of biological samples, such asproteins, nucleic acids, etc. Albumin proteins, such as bovine serumalbumin (BSA) are examples of proteins that can be used to coat amicrofabricated filter of the present invention. Polymers used to coat afilter can be any polymer that does not promote cell sticking to thefilter, for example, nonhydrophobic polymers such as, but not limitedto, polyethylene glycol (PEG), polyvinylacetate (PVA), andpolyvinylpyrrolidone (PVP), and a cellulose or cellulose-likederivative.

A filter made of, for x example, metal, ceramics, a polymer, glass, orsilica can be coated with a compound by any feasible means, such as, forexample, adsorption or chemical conjugation.

In many cases, it can be advantageous to surface-treat the filter priorto coating with a compound or polymer. Surface treatment can increasethe stability and uniformity of the coating. For example, a filter canbe treated with at least one acid or at least one base, or with at leastone acid and at least one base which can be applied in either order,prior to coating the filter with a compound or polymer. In preferredaspects of the present invention, a filter made of a polymer, glass, orsilica is treated with at least one acid and then incubated in asolution of the coating compound for a period of time ranging fromminutes to days. For example, a glass filter can be incubated in acid,rinsed with water, and then incubated in a solution of BSA, PEG, or PVP.

In some aspects of the present invention, it can be preferred to rinsethe filter, such as in water (for example, deionized water) or abuffered solution before acid or base treatment or treatment with anoxidizing agent, and, preferably again before coating the filter with acompound or polymer. Where more than one type of treatment is performedon a microfabricated filter, rinses can also be performed betweentreatments, for example, between treatment with an oxidizing agent andan acid, or between treatment with an acid and a base. A filter can berinsed in water or an aqueous solution that has a pH of between about3.5 and about 10.5, and more preferably between about 5 and about 9.Non-limiting examples of suitable aqueous solutions for rinsing iontransport measuring means can include salt solutions (where saltsolutions can range in concentration from the micromolar range to 5M ormore), biological buffer solutions, cell media, or dilutions orcombinations thereof. Rinsing can be performed for any length of time,for example from minutes to hours.

The concentration of a compound or polymer solution used to coat afilter can vary from about 0.02% to 20% or more, and will depend in parton the compound used. The incubation in coating solution can be fromminutes to days, and preferably is from about 10 minutes to two hours.

After coating, the filter can be rinsed in water or a buffer.

In one preferred embodiment of the present invention the filter has adiameter of 50 to 99.9% of the diameter of the filtration chamber. Thespace located between the edge of the filter and the walls of thefiltration chambers is occupied by, for example, an O-ring or similarsealing member that is attached to the outer edge of the filter. TheO-ring provides a tight seal between the filter and the wall of thefiltration chamber to prevent cells from bypassing the filter duringoperation.

In the present invention the filter can be positioned at differentheights inside the filtration chamber, by means of positive or negativepressure applied to a piston, which is connected to the filter.

A method for debulking a biological sample, for example a blood samplecontaining cancer cells, utilizing an automated instrument containingthe french press filtration chamber of the present invention is outlinedbelow:

A sample of 10 mL of blood, stored at room temperature for up to 4 days,is transferred to a 50 mL centrifugation tube and the volume is adjustedto 30 mL with a solution consisting of: 5 mM EDTA, 1% BSA in HBSS, PBS.A conduit automatically transfers the 30 mL of solution sample to theline connected to the inlet of the filtration chamber. After filling ofthe filtration chamber with the fluid sample, the valve controlling flowthrough the inlet is closed. Positive pressure is applied on the piston,which lowers the position of the filter (FIG. 1, B). Positive pressureon the piston is adjusted to reach a filtration rate of the fluid sampleof 0.1 to 50 mL per minute. The filter is lowered until 80 to 95% of thefluid in the filtration chamber is above the filter. The solution belowthe filter contains the cancer cells and other non-cancer cells andconstitutes the de-bulked sample.

The surface treatment methods of the present invention can also beapplied to components to be used for manipulation of biological samplessuch as chips other than those that comprise pores for filtration. Forexample, chips that comprise metals, ceramics, one or more polymers,silicon, silicon dioxide, or glass can be physically or chemicallytreated using the methods of the present invention. Such chips can beused, for example, in separation, analysis, and detection devices inwhich biological species such as cells are separated, detected, oranalyzed. The treatment of the chip can enhance or reduce theinteraction of the cells with the chip surface, depending of thetreatment used, the properties of the cells being manipulated, and thenature of the manipulation. For example, coating the surface of the chipwith a hydrophilic polymer (for example but not limited to coating thechip with PVP or PVA) may reduce or minimize the interaction between thesurface of the chip and the cells.

In some preferred embodiments of the present invention the cellsobtained from the enrichment procedures can be detected, analyzed ormanipulated by means of active force chips.

In some preferred embodiments, traveling-wave dielectrophoretic forcescan be generated by electrodes built onto a chip and can be used to movecells to different compartments. A full description of the travelingwave dielectrophoresis is provided in U.S. application Ser. No.09/679,024, entitled “Apparatuses Containing Multiple Active ForceGenerating Elements and Uses Thereof” filed Oct. 4, 2000, hereinincorporated by reference in its entirety.

One problem encountered with a filtration device such as that depictedin FIG. 1 is that cells in the sample may enter or even plug the holesin the filter as the filter advances into the sample. This can result inloss of or damage to the cells, or in plugging of the filter. One aspectof the present invention addresses this problem by providing means torepel cells from the filter surface. The following discussion andreferences can provide a framework for the design and use of electrodesto facilitate filtration by translocating sample components, such asnonfilterable cells, away from a filter using a repulsive force.

Dielectrophoresis refers to the movement of polarized particles in anon-uniform AC electrical field. When a particle is placed in anelectrical field, if the dielectric properties of the particle and itssurrounding medium are different, the particle will experiencedielectric polarization. Thus, electrical charges are induced at theparticle/medium interface. If the applied field is non-uniform, then theinteraction between the non-uniform field and the induced polarizationcharges will produce net force acting on the particle to cause particlemotion towards the region of strong or weak field intensity. The netforce acting on the particle is called dielectrophoretic force and theparticle motion is dielectrophoresis. Dielectrophoretic force depends onthe dielectric properties of the particles, particle surrounding medium,the frequency of the applied electrical field and the fielddistribution.

Traveling-wave dielectrophoresis is similar to dielectrophoresis inwhich the traveling-electric field interacts with the field-inducedpolarization and generates electrical forces acting on the particles.Particles are caused to move either with or against the direction of thetraveling field. Traveling-wave dielectrophoretic forces depend on thedielectric properties of the particles and their suspending medium, thefrequency and the magnitude of the traveling-field. The theory fordielectrophoresis and traveling-wave dielectrophoresis and the use ofdielectrophoresis for manipulation and processing of microparticles maybe found in various publications (e.g., “Non-uniform SpatialDistributions of Both the Magnitude and Phase of AC Electric Fieldsdetermine Dielectrophoretic Forces by Wang et al., in Biochim BiophysActa Vol. 1243, 1995, pages 185-194”, “Dielectrophoretic Manipulation ofParticles” by Wang et al, in IEEE Transaction on Industry Applications,Vol. 33, No. 3, May/June, 1997, pages 660-669, “Electrokinetic behaviorof colloidal particles in traveling electric fields: studies using yeastcells” by Huang et al, in J. Phys. D: Appl. Phys., Vol. 26, pages1528-1535, “Positioning and manipulation of cells and microparticlesusing miniaturized electric field traps and traveling waves” By Fuhr etal., in Sensors and Materials. Vol. 7: pages 131-146, “Dielectrophoreticmanipulation of cells using spiral electrodes” by Wang, X-B. et al., inBiophys. J. Volume 72, pages 1887-1899, 1997, “Separation of humanbreast cancer cells from blood by differential dielectric affinity” byBecker et al, in Proc. Natl. Acad. Sci., Vol., 92, January 1995, pages860-864).

The manipulation of microparticles with dielectrophoresis and travelingwave dielectrophoresis include concentration/aggregation, trapping,repulsion, linear or other directed motion, levitation, separation ofparticles. Particles may be focused, enriched and trapped in specificregions of the electrode reaction chamber. Particles may be separatedinto different subpopulations over a microscopic scale. Relevant to thefiltration methods of the present invention, particles may betransported over certain distances. The electrical field distributionnecessary for specific particle manipulation depends on the dimensionand geometry of microelectrode structures and may be designed usingdielectrophoresis theory and electrical field simulation methods.

The dielectrophoretic force F_(DEP z) acting on a particle of radius rsubjected to a non-uniform electrical field can be given by

F _(DEP z)=2π∈_(m) r ³χ_(DEP) ∇E _(rms) ² ·{right arrow over (a)} _(z)

where E_(rms) is the RMS value of the field strength, ∈_(m) is thedielectric permitivity of the medium. χ_(DEP) is the particle dielectricpolarization factor or dielectrophoresis polarization factor, given by

${\chi_{DEP} = {{Re}( \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} - {2ɛ_{m}^{*}}} )}},$

“Re” refers to the real part of the “complex number”. The symbol

$ɛ_{x}^{*} = {ɛ_{x} - {j\frac{\sigma_{x}}{2\pi \; f}}}$

is the complex permitivity (of the particle x=p, and the medium x=m).The parameters ∈_(p) and σ_(p) are the effective permitivity andconductivity of the particle, respectively. These parameters may befrequency dependent. For example, a typical biological cell will havefrequency dependent, effective conductivity and permitivity, at least,because of cytoplasm membrane polarization.

The above equation for the dielectrophoretic force can also be writtenas

F _(DEP z)=2π∈_(m) r ³χ_(DEP) V ² P(z){right arrow over (a)} _(z)

where p(z) is the square-field distribution for a unit-voltageexcitation (V=1 V) on the electrodes, V is the applied voltage.

There are generally two types of dielectrophoresis, positivedielectrophoresis and negative dielectrophoresis. In positivedielectrophoresis, particles are moved by dielectrophoresis forcestowards the strong field regions. In negative dielectrophoresis,particles are moved by dielectrophoresis forces towards weak fieldregions. Whether particles exhibit positive or negativedielectrophoresis depends on whether particles are more or lesspolarizable than the surrounding medium. In the filtration methods ofthe present invention, electrode patterns on one or more filters of afiltration chamber can be designed to cause sample components such ascells to exhibit negative dielectrophoresis, resulting in samplecomponents such as cells being repelled away from the electrodes on thefilter surfaces.

Traveling-wave DEP force refers to the force that is generated onparticles or molecules due to a traveling-wave electric field. Atraveling-wave electric field is characterized by the non-uniformdistribution of the phase values of AC electric field components.

Here we analyze the traveling-wave DEP force for an ideal traveling-wavefield. The dielectrophoretic force F_(DEP) acting on a particle ofradius r subjected to a traveling-wave electrical field E_(TWD)=Ecos(2π(ft−z/λ₀)){right arrow over (a)}_(x) (i.e., a x-direction field istraveling along the z-direction) is given by

F _(TWD)=−2π∈_(m) r ³ζ_(TWD) E ² ·{right arrow over (a)} _(z)

where E is the magnitude of the field strength, ∈_(m) is the dielectricpermittivity of the medium. ζ_(TWD) is the particle polarization factor,given by

${\zeta_{TWD} = {{Im}( \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} - {2ɛ_{m}^{*}}} )}},$

“Im” refers to the imaginary part of the “complex number”. The symbol

$ɛ_{x}^{*} = {ɛ_{x} - {j\frac{\sigma_{x}}{2\pi \; f}}}$

is the complex permittivity (of the particle x=p, and the medium x=m).The parameters ∈_(p) and σ_(p) are the effective permittivity andconductivity of the particle, respectively. These parameters may befrequency dependent.

Particles such as biological cells having different dielectric property(as defined by permittivity and conductivity) will experience differentdielectrophoretic forces. For traveling-wave DEP manipulation ofparticles (including biological cells), traveling-wave DEP forces actingon a particle of 10 micron in diameter can vary somewhere between 0.01and 10000 pN.

A traveling wave electric field can be established by applyingappropriate AC signals to the microelectrodes appropriately arranged ona chip. For generating a traveling-wave-electric field, it is necessaryto apply at least three types of electrical signals each having adifferent phase value. An example to produce a traveling wave electricfield is to use four phase-quardrature signals (0, 90, 180 and 270degrees) to energize four linear, parallel electrodes patterned on thechip surfaces. Such four electrodes form a basic, repeating unit.Depending on the applications, there may be more than two such unitsthat are located next to each other. This will produce atraveling-electric field in the spaces above or near the electrodes. Aslong as electrode elements are arranged following certain spatiallysequential orders, applying phase-sequenced signals will result inestablishing traveling electrical fields in the region close to theelectrodes.

Both dielectrophoresis and traveling-wave dielectrophoresis forcesacting on particles depend on not only the field distributions (e.g.,the magnitude, frequency and phase distribution of electrical fieldcomponents; the modulation of the field for magnitude and/or frequency)but also the dielectric properties of the particles and the medium inwhich particles are suspended or placed. For dielectrophoresis, ifparticles are more polarizable than the medium (e.g., having largerconductivities and/or permitivities depending on the applied frequency),particles will experience positive dielectrophoresis forces and aredirected towards the strong field regions. The particles that are lesspolarizable than the surrounding medium will experience negativedielectrophoresis forces and are directed towards the weak fieldregions. For traveling wave dielectrophoresis, particles may experiencedielectrophoresis forces that drive them in the same direction as thefield traveling direction or against it, dependent on the polarizationfactor ζ_(TWD). The following papers provide basic theories andpractices for dielectrophoresis and traveling-wave-dielectrophoresis:Huang, et al., J. Phys. D: Appl. Phys. 26:1528-1535 (1993); Wang, etal., Biochim. Biophys. Acta. 1243:185-194 (1995); Wang, et al., IEEETrans. Ind. Appl. 33:660-669 (1997).

Thus a filter for use in the methods described herein can advantageouslyinclude one or more electrodes such as the patterned electrodesdescribed above. The electrode(s) may be on a surface of the filter fora press such as the one depicted in FIG. 1; typically, one or moreelectrodes would be placed on the surface of the filter facing thesample to be debulked, and the electrodes would be turned on before andoptionally during the time period in which the filter is advanced intothe sample. However, the electrode(s) may also be contained within amaterial used to construct the filter or on the face of the filtrationmaterial from the sample, provided the electrodes produce sufficientdielectrophoretic force on cells in the sample to be debulked so that atleast some of the cells, preferably including the target cells, arerepelled from the filter. The electrodes thus repel cells from thefilter surface and permit filtration to be more effective and lesslikely to cause damage to or loss of the target cells.

Various combinations of the above enrichment methods can of course beused, as those skilled in the art will appreciate, depending upon thevolume and nature of the sample to be enriched, and depending upon thetype of cells to be enriched and to be removed. The target cellsobtained at the end of the enrichment protocol described in the presentinvention can be further purified from other non-target cells present inthe sample by an additional positive selection step, or they may becharacterized before further enrichment occurs.

In many embodiments of the present invention, the target cells areisolated as intact, viable cells that can be grown in culture forfurther characterization and use. In one preferred embodiment of thepresent invention, cancer cells obtained by enrichment methods such asthose described herein are grown in culture in artificial nutrientsfollowing their enrichment from a cancer patient blood sample. Thispermits the user to identify a preferred therapeutic protocol based onthe specific cells to be targeted by observing how the particular cellsreact to various proposed treatments, for example, or to use theenriched cancer cells in other ways such as in screening drug candidatesto determine which types of cancers they are likely to treat.

A method for growing tumor cells obtained from a blood sample isoutlined below. A sample of 10 mL of blood, stored at room temperaturefor up to 4 days, is transferred to a 50 mL centrifugation tube and thevolume is adjusted to 30 mL with a solution consisting of: 5 mM EDTA, 1%BSA in HBSS, and PBS or HBSS. The sample is centrifuged at 1400 rpm for5 min at room temperature. 23 mL of supernatant are discarded and thepellet is completely resuspended by gentle shaking. An RBC lysis bufferis added (22 mL), and the sample is incubated with rotation at roomtemperature for about 8 mM Following the lysis of the red blood cellsthe sample is centrifuged at 1400 rpm for 5 mM at room temperature. Thesupernatant is aspirated, leaving the remaining cell pellet undisturbed.The cell pellet is completely resuspended in 45 mL of solutioncontaining 5 mM EDTA, 1% BSA in HBSS, and PBS. The sample is centrifugedagain at 1400 rpm for 5 mM at room temperature; the supernatant isdiscarded and the cells are resuspended in 0.3 mL of solution containing5 mM EDTA, 1% BSA in HBSS, and PBS. The cell suspension is transferredinto a 2 ml (U-bottom) eppendorf tube and 0.8 mL of AVIVA beads slurryis added. The beads are coated with antibody recognizing the CD50antigen. The cells/beads suspension is incubated with gentle rotationfor 20 minutes at room temperature. Following the incubation, the tubesare positioned on a magnetic stand at room temperature for 1 mM withlead cap open, to allow for the beads and the non-cancer cells adsorbedto the beads to migrate towards the side of the tube facing the magnet.The solution is carefully transferred to a 1.7 mL eppendorf tube(V-bottom). The enriched cells are centrifuged at 10,000 rpm for 1minute. After discarding the supernatant the cells are resuspended in0.5 mL of growth media containing RPMI-1640, and DMEM-High Glucose. Thecells suspension is then transferred to a microtiter plate or artificialgrowth matrix and incubated at 37° C. and 5% CO₂ to allow cell growth.

In some preferred embodiments of the present invention the culturedcancer cells isolated from a cancer patient are used as a substrate fortesting the anti-cancer activity of drugs. This enables the user toidentify a suitable chemotherapy protocol for the particular cancer. Amethod for testing the anticancer activity of drugs on cancer cells froma cancer patient is outlined below:

One or more cancer cells isolated from a cancer patient utilizing themethod of the present invention are aliquoted into multiple separatedcompartments in a microtiter plate. The cells are grown for anappropriate amount of time from about 1 hour to 50 days, preferably 124to 336 hours. Following the growth period, one specific anti-cancercandidate, which may be a single drug or a mixture of drugs, is added ineach compartment containing the cancer cells, and at least onecompartment contains a control solution which does not contain drugs.Cells are grown in the presence of the anti-cancer candidate(s) for anappropriate amount of time from about 1 hour to 5 days. The anti-cancercandidates are scored for their ability to induce cellular changes,which may take the form of: apoptosis, necrosis, cytotoxicity, reducedstemness, cell death, inhibition of cell growth, and/or inhibition ofcell division of the cancer cells.

To those skilled in the arts it will be evident that the anti-cancercandidate can be tested at several different concentrations on thesecultured cancer cells or on cells actually isolated from a cancerpatient, in order to provide guidance on the dosage of the drug to beadministered to the patient.

II. Method of Detection of Cancer Cells Obtained from a Blood Sample.

Once a sample has been enriched with a target non-hematopoietic celltype, the presence, absence and/or amount of the enriched target cell inthe sample, e.g., a non-hematopoietic cancer cell, can be assessed by anumber of methods. In one example, the presence, absence and/or amountof the enriched non-hematopoietic cell or tumor cell can be assessed bylabeling the enriched non-hematopoietic cell or tumor cell andidentifying the labeled non-hematopoietic cell or tumor cell. Theenriched non-hematopoietic cell or tumor cell can be labeled using anysuitable methods, e.g., immunostaining, DNA content measurement, in situPCR, in situ hybridization, fluorescence in situ hybridization (FISH),staining by a labeled binding member that specifically binds to theenriched non-hematopoietic cell or tumor cell. The labelednon-hematopoietic cell tumor cell can be identified and characterized byany suitable methods, e.g., microscopic analysis, and/or flowcytometryas well as laser based quantitative microscopy technology such as laserscanning cytometry. In one specific embodiment, the labelednon-hematopoietic cell, or tumor cell, is identified and characterizedby cell counting, after the target cells have been labeled, typicallyusing one or more immunostaining methods.

Labeling of the target cells is often accomplished with an immunologicallabeling method, such as attaching a fluorescence label to the cellsusing an antibody specific for a marker present on the surface of thetarget cells. The label can be linked to the antibody, or a firstantibody specific for the targeted cells can be allowed to bind to thecells, and subsequently the first antibody can be labeled by allowing asecond labeled antibody that is specific for the first antibody to bindto the first antibody that is bound to the target cells. To clearlyidentify the target cells, more than one labeling process may be used.For example, the cells may be labeled with a first labeling method thatidentifies the target cell as a non-hematopoietic cell and with a secondantibody that identifies the cell as a mutated cell, or as a cell from aparticular tissue, etc. A combination of labeling processes may includenegative and/or positive labeling processes, i.e., labels thatspecifically bind to the target cells would be considered a positivelabeling process, while labels that specifically bind to non-targetcells and allow them to be distinguished from the target cells would beconsidered a negative labeling process. Thus a set of two or morelabeling methods may be used to label the target cell and/or other cellspresent to unambiguously distinguish the target cell as a cancer cell,for example.

A critical aspect of the present invention is the selection of aplurality of antigens that are used as selective target cells markersand allow for the identification of target cells, such as cancer cells,with increased specificity, and in particular for distinguishing acancer cell from a non-cancerous circulating epithelial cell in a bloodsample, for example. In the present invention combinations of two ormore of the following markers can be used for differentiating betweencancer cells enriched from a cancer patient blood sample and thebackground of non-neoplastic cells: ACPP, AFP, albumin, ALCAM, AMAS,ARF6, ARMCX3, ATP1A1, BAG1, BJ-TSA-9, blc-2 βHCG, CA125, CA15-3, CA19-9,Cathepsin B1, CD44, CD44v6, CD56, CD66a, CD66b, CD66c, CD66d, CD66e,CD66f, CD147, CDH2, CDK4I, CDKN2A, CDX2, CEA, CLDN3, CLDN4, CLDN5,c-met, CST3, Cytokeratins, CK18, CK19, CK20, Desmoplakin-3, EAG1, EGFR,EGP2, EMA, ErbB2, ESR1, FAK, FOXA2, GalNac-T, GCTFTI5, GFAP,Haptoglobin-α, HCA, hCASK, HE4, HEPA1, hERG, HIP-1, HMB45, HSPA2, IGFR,IVL, KCNK-9, KHDRBS3, Ki67, Kv1.3, LAMB2, Lewis-Y antigen, LIMA, LMO6,LUNX, MAGE-3, MAGE-A3, mammoglobin, Maspin, Melan-A, MITF, MPP5, MPST,MUC-1, MUC5AC, NCAM-1, NSDHL, Oct4, OTC, p53, p97, p1B, PCNA, PGR, PMSA,PS-2, PSA, RPS6KA5, S100, S100A1, S100A2, S100B, SLC2A1, Smoothelin,SP-1, SPARC, Surfactant, Telomerase, TFAP2A, TITF1 (TTF1), TFF2, TRAIL,TRIM28, TRPM-8, TYR, Tyrosinase, TYRP1, Ubiquitin thiolesterase, VEGF,WT1, X-protein, ZNF165. In the embodiments where a multiplicity ofspecific binding members is used, each binding member might carry none,the same, or different labels. Using suitable combinations of two ormore of such markers, cancerous cells can be distinguished from normalepithelial cells that may be present with at least 50% reliability, andpreferably with at least about 70% reliability. In certain embodiments,the combination of markers distinguishes a cancer cell from a normalepithelial cell at least 80% of the time, which provides suitablespecificity to enhance the diagnostic value of the methods.

The cancer cell enrichment method of the present invention can provide asample in which a mixture of cancer cells and non-cancer cells arepresent. Final and unequivocal identification of the cancer cells, whichcan include enumeration of total cancer cells, identification of tissueorigin of the cells, and/or genotypic characterization of the cells, isoften achieved by labeling of said cancer cells. The specific labelingof the cancer cells can be achieved using suitable immunostainingmethods well known to those individuals versed in the arts. As anexample of a suitable procedure, the cells can be stained as outlinedbelow:

A sample of enriched cells is transferred to a surface and air-dried.This may be done by centrifugation of the enriched sample to provide acell pellet, which is then transferred to a suitable surface foranalysis, such as a plastic or glass microscope slide. The cells arethen washed once with 20 mL of PBS for 5 min and subsequently fixed with2% PFA (paraformaldehyde) in PBS for 40 min at room temperature. Afterrinsing with PBS three times, the cells are permeabilized by incubationin 0.1% Triton X-100 dissolved in PBS for 5 min. The cells are thenrinsed three times with PBS for 5 minutes. Cells are incubated for 1 hrat room temperature in 0.2% BSA-PBS with monoclonal antibodies againstone or more of a selected list of markers at dilutions of 1:50-1:100.After rinsing three times for 5 min, the cells are incubated withAlexa-labeled secondary antibody, in 0.2% BSA-PBS at room temperaturefor 1 hr in the dark. After rinsing the glass slides three times with0.2% BSA and PBS for a total of 10 minutes, the cells are stained withHematoxylin for 1 min. Cells are subsequently rinsed in PBS for 1 min,changing PBS twice, and mounted with mounting medium (10 μL).

In one embodiment of the present invention, following the enrichmentprotocol the cancer cells are positively identified as cells labeled bybinding members recognizing one, or two, or three or more of the markerslisted below, and optionally also by specific morphological criteriarelated to the diameter of its nucleus, shape of the nucleus, diameterof the whole cell, or the ratio of the cytoplasmic portion of the cellsto its nucleus. The cancer cell marker may include one or more of thefollowing: ACPP, AFP, albumin, ALCAM, AMAS, ARF6, ARMCX3, ATP1A1, BAG1,BJ-TSA-9, blc-2 βHCG, CA125, CA15-3, CA19-9, Cathepsin B1, CD44, CD44v6,CD56, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD147, CDH2, CDK4I,CDKN2A, CDX2, CEA, CLDN3, CLDN4, CLDN5, c-met, CST3, Cytokeratins, CK18,CK19, CK20, Desmoplakin-3, EAG1, EGFR, EGP2, EMA, ErbB2, ESR1, FAK,FOXA2, GalNac-T, GCTFTI5, GFAP, Haptoglobin-α, HCA, hCASK, HE4, HEPA1,hERG, HIP-1, HMB45, HSPA2, IGFR, IVL, KCNK-9, KHDRBS3, Ki67, Kv1.3,LAMB2, Lewis-Y antigen, LIMA, LMO6, LUNX, MAGE-3, MAGE-A3, mammoglobin,Maspin, Melan-A, MITF, MPP5, MPST, MUC-1, MUC5AC, NCAM-1, NSDHL, Oct4,OTC, p53, p97, p1B, PCNA, PGR, PMSA, PS-2, PSA, RPS6KA5, S100, S100A1,S100A2, S100B, SLC2A1, Smoothelin, SP-1, SPARC, Surfactant, Telomerase,TFAP2A, TITF1 (TTF1), TFF2, TRAIL, TRIM28, TRPM-8, TYR, Tyrosinase,TYRP1, Ubiquitin thiolesterase, VEGF, WT1, X-protein, ZNF165. Instead ofor in addition to the above markers, morphological criteria used tofurther identify the cancer cells include the following: a) cell nucleushaving a diameter larger than a statistically pre-determined value, b)cytoplasm diameter to nucleus diameter ratio between 1.01 and 20, c)overall cell diameter between 3 and 50 uM.

One important difference between normal cells and cancer cells is thatnormal cells only divide for a limited number of times while cancercells can continue to divide almost indefinitely. The aging of normalcells is not determined by chronological time but a sort of internalclock which tracks the number of cell divisions. Evidence hasaccumulated in recent years that one way cells track the number of celldivision is the progressive shortening of the chromosomes ends(telomeres) which is a byproduct of each cell division. Telomerase is anenzyme capable of restoring the length of telomeres and can “reset” thebiological clock, which measures cell age. In normal cells, however, theexpression and activity of telomerase are extremely low, which leads tocell senescence after a finite number of cell divisions. On thecontrary, in the majority of cancers, the cells express significantamounts of telomerase and the high level of telomerase activity allowsthese cells to maintain a constant length of their telomeres and stopthe clock that measures aging.

In one aspect of the present invention, the presence or the growth andmetastatic potential of cancer cells isolated from the blood of cancerpatients is measured by determining the level of expression and/or thefunction of telomerase in said cancer cells. The enrichment of thecancer cells from the patient sample prior to performing the telomerasedetection can significantly improve the sensitivity of the assay. Thecancer cells can be isolated from a patient sample, like a blood sample,following a positive selection method like the one described by Griwatzand colleagues (Griwatz C., Brandt B., Assmann G., Zanker K. S., Journalof Immunological Methods, 183 (1995) 251-265). Other enrichment methodsknown to those skilled in the arts can also be used to isolate thecancer cells from the patient sample, prior to performing telomerasedetection; or the sample containing the cells can be enriched by themethods described herein. Optionally, enrichment by methods such asthose described herein can be combined with a positive selection step.Following the isolation of the cancer cells or the enrichment of thesample, telomerase can be detected in the cancer cells or enrichedsample by using a telomerase activity assay like telomerase repeatamplification protocol (TRAP) or using immunodetection methods based onantibodies or other ligands with specific affinity for telomerase todetect the protein itself. The level of telomerase or of its activity inthe sample, or the level of a telomerase-encoding nucleic acid or thelevel of the expression of such nucleic acid in the sample, can be usedto determine the probability that the sample contained a cancer cell, orto predict the aggressiveness of the tumor or its likelihood ofmetastasis, or to determine the number or proportion of cancer cells inthe blood sample.

In one preferred embodiment of the present invention the presence or thegrowth and metastatic potential of cancer cells isolated from the bloodof cancer patients is measured by determining the level of expressionand/or the function of telomerase in said cancer cells. The cancer cellsare isolated from a patient's sample using the depletion methodsdescribed in the present invention. Following the isolation or theenrichment of the cancer cells telomerase can be detected in the cancercells in the sample using telomerase activity assay like telomeraserepeat amplification protocol (TRAP) or immunodetection methods based onantibodies or other ligands with specific affinity for telomerase. Thelevel of telomerase activity or of telomerase expression in the cancercells in the sample, can be an indicator of the aggressiveness of thetumor or of the number or proportion of cancer cells in the enrichedsample.

The glycoprotein CD44 is a cell adhesion molecule originally discoveredin lymphocytes and granulocytes but is fairly ubiquitously expressedthroughout the body. Up regulation of CD44 and in particular of splicevariants (CD44v) like CD44v6, CD44v7, CD44v8, CD44v6-10 is typical ofmany cancers.

In another embodiment of the present invention the presence or thegrowth and metastatic potential of the cancer cells isolated from theblood of cancer patients is measured by determining the level ofexpression and/or function of one or more CD44v splice variants in saidcancer cells.

One or more cancer markers can be assessed. In one example, a singlecancer cell marker is assessed. In another example, a plurality of thecancer cell markers is assessed. The plurality of the markers can bederived from the same or different cells, e.g., different cell markersare detected simultaneously, e.g., using a high-throughput assay.

In one embodiment of the present invention following the enrichment ofthe cancer cells from a blood sample, the cancer cells are identified bylabeling with monoclonal antibodies recognizing one or moreCytokeratins, CD44v6 or other CD44 splice variants, and Telomerase.

In another example, the presence, absence and/or amount of the enrichednon-hematopoietic cell or cancer cell can be assessed by identifyingnucleic acids such as mRNA of the enriched non-hematopoietic cell ortumor cell. Any suitable methods, especially nucleic acid polymerasebased methods, can be used to identify the enriched cell ornon-hematopoietic tumor cell. For example, the presence, absence and/oramount of the enriched non-hematopoietic cell or cancer cell can beassessed by PCR. Any suitable PCR methods can be used. (See e.g.,Singleton and Sainbury, DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY, Third ed., pages 557-560). In one specific embodiment,singleplexed or multiplexed RT-PCR can be used. In another specificembodiment, qRT-PCR or Real Time PCR can be used.

In addition to cell identification analysis, the enrichednon-hematopoietic cancer cell can be subjected to additional analysis.Any suitable methods can be used. For example, PCR, RNA-basedamplification, oligonucleotide ligation assay (OLA), laser dissectionmicroscopy (LDM), whole genome amplification (WGA), comparative genomichybridization (CGH), DNA methylation assay, microarray analysis, totalDNA content or a combination thereof can be used in the additionalanalysis. In one specific embodiment, the additional analysis comprisesassessing DNA methylation of the enriched non-hematopoietic cancer cell.(Diala et al., J. Natl. Cancer Inst., 71(4):755-64 (1983); Frost et al.,Cancer Metastasis. Rev., 2(4):375-8 (1983); and Weber et al., NatureGenetics, 37(8):853-62 (2005)).

The present methods can be used for detecting any suitablenon-hematopoietic cancer cell in a blood sample. For example, thepresent methods can be used for detecting a solid tumor cell in a bloodsample. Exemplary tumors include hemangioendothelioma, apudoma,choristoma, branchioma, malignant carcinoid syndrome, carcinoid heartdisease, carcinoma e.g., Walker, basal cell, basosquamous Ehrlich tumor,merkel cell, mucinous, non-small cell lung, oat cell, papillary,scirrhous, bronchiolar, bronchogenic, squamous cell and transitionalcell reticuloendotheliosis, melanoma, chondroblastoma, chondroma,chondrosarcoma, fibroma, fibrosarcoma, myosarcoma, giant cell tumors,histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma,osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma,adenolymphoma, carcinosarcoma, chordoma, mesenchymoma, mesonephroma,myosarcoma, cementoma, odontoma, teratoma, throphoblastic tumor,adenocarcinoma, adenoma, cholangioma, angiomatosis, cholesteatoma,paraganglioma nonchromaffin, cylindroma, cystadenocarcinoma,cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma,hidradenoma, islet cell tumor, leydig cell tumor, papilloma, sertolicell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma,myoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma,glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma,neuroepithelioma, neurofibroma, neuroma, paraganglioma, antiokeratoma,angioma sclerosing, glomangioma, hemangioma, hemangiopericytoma,hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma,pinealoma, carcinosarcoma, chondrosarcoma, ameloblastoma, cystosarcomaphyllodes, fibrosarcoma, Brown-Pearce, ductal, hemangiosarcoma,leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma,myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma(Kaposi's, and mast-cell), neoplasms (e.g., bone, digestive system,colorectal, liver, pancreatic, pituitary, testicular, orbital, head andneck, central nervous system, acoustic, pelvic, respiratory tract, andurogenital), neurofibromatosis, and cervical dysplasia.

EXAMPLES Example 1 Enrichment and Analysis of Cancer Cells from a BloodSample

Materials and Methods

Affinity-purified anti-CD50 monoclonal antibody was obtained from AvivaSystem Biology (San Diego, Calif.). Normal human blood collected inanti-coagulant ACD tubes were provided by Advanced Biosciences Resource(Alameda, Calif.). NHS Biotin and DAPI were purchased from Pierce(Rockford, Ill.) and Invitrogen (Carlsbad, Calif.), respectively. Avidincoated magnetic beads were generated in house. FITC-anti-CD31 andPE-anti-CD50 mAbs were from Ancel.

Examination of CD50 and CD31 Expression on Carcinoma Cell Lines by FACS

Carcinoma cells from a series of cell lines including human breastcancer (MDA-4355), human colon cancer (DLD-1), human prostate cancer(PC-3), and human cervix cancer (HeLa) were trypsinized, followed byspinning down at 1300 rpm for 5 min. Cells were resuspended in 1%BSA-PBS and aliquoted into a polystyrene FACS tube (Falcon, Product#352054) to have 1×10⁶ cells/0.1 ml/tube Immunofluorescence dyeconjugated mAbs including anti-CD50, anti-CD31 and control antibodieswere added (1 ug/tube), followed by incubation on ice for 30 min Cellswere washed twice with 1% BSA-PBS at 1300 rpm for 3 min, followed byfixation in 2% paraformaldehyde in PBS, 0.5 ml/tube. Samples were readyfor FACS analysis.

Biotinylatyion of Anti-CD50 mAb

Anti-CD50 mAb were dialyzed against PBS buffer at 4° C. overnight. Forbiotin labeling of antibody, 5-30 fold molar of excess biotin wereincubated with IgG at 4° C. overnight with rotation. ConjugatedBiotin-IgG were quantified using BCA protein assay kit (Pierce).

Coating Biotinylated IgG to the Avidin-Magnetic Beads

Avidin-magnetic beads were washed four times with PBE (PBS containing0.5% BSA and 10 mM EDTA, pH 7.4) using a magnetic stand, followed byincubation with biotin labeled IgG (5-150 ug biotin-Ab/10⁹ beads) at 4°C. for 30 min with rotation. Beads were washed four times with PBE toget rid of unconjugated biotin-IgG, and stored in PBE buffer with thestarting volume to get 1×10⁹ beads/ml.

Tumor Cell Enrichment (Negative Depletion)

For all cell spiking studies, spiked with two cells, live carcinomacells in suspension were labeled with DAPI dissolved in culture mediafor at least 24 hours. Exact desired number of labeled cells were pickedup by means of a micromanipulator (Sutter Instruments) under amicroscope. For spiking studies with more than two cells, cells werefixed, permeabilized, and labeled with DAPI, followed by quantificationusing a hemocytometer. Desired number of cells were estimated by mixinga known number of cells in PBS followed by limited dilution, spiked into10 ml human blood in 50 ml tube, followed by adjustment of volume to 45ml with PBE. Blood samples were centrifuged at 1400 rpm for 5 min. Thesupernatants were removed by aspiration. Forty five ml of red blood celllysis buffer were added into sedimentary cells and subsequently rotatedat room temperature for 8 min. The samples were spun down at 1400 rpmfor 5 min Cell pellets containing WBC and rare cells were washed oncewith 45 ml PBE at 1400 rpm for 5 min and resuspended in 0.3 ml of PBE.Meanwhile, anti-CD50 beads were washed 3 times with a magnetic stand, 1ml/wash, and subsequently resuspended in 0.8 ml of PBE. Aboveresuspended cells were mixed with washed anti-CD50 beads, followed byincubation at room temperature for 30 min with rotation. The sample tubewas subsequently put in a magnetic stand for 1 min. The supernatant wascompletely transferred into an eppendorf tube, followed bycentrifugation at 14,000 rpm for 1 min. The supernatant was aspirated,and the remaining cells were resuspended in 10 ul of mounting media, andsubsequently subjected to immunofluorescence analysis.

Results

Examination of CD31 and CD50 Expression on Human White Blood Cells andCarcinoma Cells

FACS analysis shows strong positive CD50 staining on human WBC includinglymphocytes monocytes and granulocytes (FIG. 2). However, humancarcinoma cells including breast cancer, colon cancer, prostate cancerand cervix cancer all show negative staining on CD50 (FIG. 3). Thepositive control Jurkat cells show positive staining on CD50, andnegative control antibodies (IgG1 and IgG2b) show negative staining onall cells (FIG. 3). These results, also summarized in the followingTables 1 and 2, indicate that anti-CD50 antibody is suitable forspecific removal of only WBCs from human blood.

TABLE 1 CD50 staining on human lymphocytes monocytes and granulocytesMean Value Granulocytes Monocytes Lymphocytes autofluorescence 66.1244.98 28.77 IgG2b 30.07 17.6 6.04 anti-CD50 5635.84 4911.88 1889.66

TABLE 2 Lack of CD50 staining on human carcinoma cells Breast ColonProstate Cervix Cancer Cancer Cancer Cancer Jurkat Mean Value Cell CellCell Cell Cells autofluorescence 3.9 3.98 4.07 3.28 2.59 IgG2b 7.2310.34 10.35 5.1 3.87 anti-CD50 13.19 16.26 14.02 6.37 182.17

Recovery of Spiked Carcinoma Cells from Human Blood

Different numbers of spiked carcinoma cells including HeLa cells,prostate cancer cells, lung cancer and breast cancer cells were isolatedfrom human blood by means of the negative depletion procedures describedherein. For HeLa cells (FIG. 4, black), following spiking with 2, 3, 5,10 cells into blood, the average recovery rate is 88%, 83%, 86%, 80% and73%, respectively. In the case of prostate cancer cells ((FIG. 4, grey),when 2 or 10 cells were added, the average recovery rate is 100% and82%, respectively. For lung cancer cells, when 2 or 10 cells were spikedinto blood, the average recovery rate was 100% and 90%. For breastcancer cells, when 2 or 10 cells were spiked into blood, the averagerecovery rate was 100% and 90%.

The negative depletion procedure illustrated above can be applied toenrich cancer cells, optionally followed by further identification andcharacterization approaches, such as antibody based positive capturing,FISH, (quantitative) RT-PCR, in situ RT-PCR, in situ hybridization,oligonucleotide ligation assay (OLA), laser dissection microscopy (LDM),whole genome application (WGA) including microarray as well ascompetitive genomic hybridization (CGH). Once circulating tumor cells(CTC) are enriched from human blood, further identification of tumorcells based upon either qRT-PCR or immunostaining of tumor cells can beperformed. For all downstreamidentification, appropriate selection of apanel of cancer markers can be different intracellular and/orextracellular cell surface markers. For instance, in the case of breastcancer, the markers can be classified into 2 different categories:tissue non-specific markers such as CK8/18, 19 and 20; EGFR, VEGF, FGF,MMP, Mucin, CD44v, beta-hCG; CEA+CK19; Maspsin etc., and tissue specificmarkers such as mammaglobulin I. The markers can further compriseapoptotic markers such as caspase 3. A panel of markers for differentcancers can be analyzed. Each panel can include both tissue specific andnon-specific markers.

Example 2 Detection of Isolated Cancer Cells by Immunofluorescence Usinga Combination of Tumor Markers

Monolayer of enriched cells on slides are immunostained with a mixtureof anti-tumor marker mAbs such as anti-CA19.9, anti-cytokeratin andanti-telomerase, labeled with fluorescent dyes, each in a differentcolor, followed by analysis using fluorescence microscopy.Alternatively, immunofluorescence can be indirectly labeled on theantibodies via biotinylation, followed by incubation with fluorescentmolecules conjugated to Avidin. In addition, immunofluorescence moleculelabeled secondary antibodies are another choice to label the unlabeledprimary antibodies recognizing target cells. An example of circulatingcancer cells from a patient blood sample identified by this method isillustrated in FIG. 5

Example 3 Detection of Isolated Cancer Cells by Immunofluorescence UsingPCR

Following the enrichment of rare cells, RNA is isolated from those cellsusing magnetic beads, column based methods, or traditional RNA isolationstrategies using guanidium, phenol/chloroform. Reverse transcription ofRNA is performed using oligo dt, gene specific and/or random primer togenerate cDNA as a template. A PCR, a qPCR or multiplex PCR issubsequently carried out to amplify specific target genes such as tumormarkers.

Reverse transcription buffer was added to the enriched sample comprisingthe target rare cells. Direct lyses of the cells was performed byheating the enriched samples at 95° C. for 1 min

Reverse transcription of RNA using oligo dT, gene specific and/or randomprimer to generate cDNA:

Resuspend above isolated lysed rare cell sample with RNA into 11 μl dNTPmixture (1 mM each) and primer (50 μM oligo(dT), 2 μM gene specificprimer and/or 50 ng/μl random hexamer). Incubate at 70° C. for 5 minutesand chill on ice. Add 5 μl reaction buffer (250 mM Tris-HCl (pH 8.3 at25° C.), 250 mM KCl, 20 mM MgCl₂, 50 mM DTT), 20u Ribonuclease inhibitor(Promega), and DEPC-treated water to total 20 μl, incubate at 42° C. for5 minutes. If random primer is used, incubate at 25° C. for 5 minutes.Add 200 units of SuperScript II (Invitrogen). Incubate the reactionmixture at 42° C. for 60 minutes. If using random hexamer primer,incubate at 25° C. for 10 minutes and then at 42° C. for 60 minutes.Terminate reaction at 85° C. for 5 minutes and chill on ice.

Amplification of specific target cDNAs by multiplex PCR:

Mix all primers together to have first run PCR (20-35 cycles), thenperform second run PCR by individual primer using aliquot from first runPCR as template (25-45 cycles). Optimize PCR conditions for eachcombination of the primers (first run and second run, MgCl₂concentration, annealing temperature, extension time, primerconcentration, and primer design). Add PCR mixture into the RT reaction,adjust volume of reaction to 50 μl using autoclaved filtered water (pH7.0). The component of PCR reaction are: 1×PCR mixture (the finalconcentration of each components are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mMMgCl₂), 200 μM dNTP, 0.4 μM of each primers, 1 unit of Taq DNApolymerase, 2% DMSO.

Example 4 Isolation and Detection of Circulating Cancer Cells fromPatients with Pancreatic Cancer

Table 3 shows the number of circulating cancer cells enriched andidentified from human subjects using the methods described in thepresent invention. Reported in the Table are the results from blindedexperiments of blood samples from 7 pancreatic cancer patients and 5control healthy subjects. In addition, the number of circulating cancercells from 2 patients with benign tumors are shown. In none of thecontrol healthy individuals was the number of circulating cancer-likecells higher than 8. In addition the 2 patients with benign tumors had 0or 1 cancer-like cell in their blood samples. In contrast, all of thepatients previously diagnosed with pancreatic cancer had >20 cancercells per sample of blood. Two patients (no. 3 and no. 6) provided bloodsamples before and after chemotherapy. For patient no. 3 the positiveeffect of chemotherapy can be seen in the drop in the number ofcirculating cancer cells (from 134 to 5), while patient no. 6 does notappear to respond favorably to the therapy. This example illustrates howthe methods described in the present invention can be used todistinguish between healthy individuals and patients with cancer and toprovide a diagnosis for metastatic cancer. It also suggests thepotential use of this method to monitor patient response to therapies.

TABLE 3 Subject # Diagnosis CTC No. 1 pancreatic cancer 20 2 pancreaticcancer 65 3 pancreatic cancer 134 3 pancreatic cancer, post chemo 5 4pancreatic cancer 21 5 pancreatic cancer 45 6 pancreatic cancer 23 6pancreatic, cancer, post chemo 34 7 pancreatic cancer 40 8 benign tumor1 8 benign tumor, different draw 0 9 benign tumor 0 10 healthy donor 111 healthy donor 1 12 healthy donor 5 13 healthy donor 4 14 healthydonor 8

Example 5 Isolation and Detection of Circulating Cancer Cells fromPatients with Lung Cancer

Table 4 reports the results obtained when the methods described in thepresent invention were used to isolate and detect cancer cells in bloodsamples from individuals with lung cancer. In this study the possibilitythat cancer-like cells could be detected in the blood as a non specificartifact resulting from the compromised general health of the patients,individuals diagnosed with tuberculosis were used as controls. All thecontrol individuals had no more than 1 cancer-like cell isolated from ablood sample, while all the patients previously diagnosed with variousforms of lung cancer (adenocarcinoma, AD; small-cell-lung cancer, SCLC),under various treatment status had 2 or more cancer cells per bloodsamples.

TABLE 4 Number of Number of Number of chemo radiation circulatingPatient Age Diagnosis Stage Metastasis Surgery treatments treatmentscancer cells Note 1 na. TB 0 2 n.a. TB 0 3 n.a. TB 0 4 n.a. TB 0 5 n.a.TB 0 6 n.a. TB 0 7 n.a. TB 0 8 n.a. TB 0 9 n.a. TB 0 10 65 Unknown 0 1177 Bladder 1 12 n.a. TB 1 13 42 Thymus 1 14 65 AC n.a. after 2 2 Relapse15 46 AC IIIA after 6 2 16 55 AC(HD) Ib after 6 2 17 40 Unknown n.a.n.a. n.a. 2 18 66 SCC IIb before 3 3 19 50 SCC IIIA n.a. 4 1 3 20 48Unknown IV Brain before 0 3 21 65 AC n.a. afer 3 4 22 37 AC IIIb after 64 23 64 AC IIIb none 5 5 2nd line 24 68 SCLC IV Lungs/Liver n.a. 4 7 2565 AC IIb after 0 8 26 n.a. Prostate n.a. n.a. n.a. 9 27 70 AC(MD) IVLungs none 2 11 28 68 SCLC n.a. n.a. 0 14 29 60 AC IIb after 0 15 30 65AC Ib before 0 16 31 50 SCC IIIA after 3 1 16 32 73 AC IV Lungs none 421 2nd line 33 71 AC n.a. n.a. 6 1 22 34 75 SCLC IIIb none 2 34 35 60 ACIIb after venal tie n.a. 57 36 72 SCLC IV Lungs n.a. 0 75 37 60 AC IIbbefore 0 85

Example 6 Comparison of Depletion Vs Selection Methods for Isolating andDetecting Cancer Cells in the Blood of Cancer Patients

In recent years a number of methods have been developed for enrichingcancer cells from blood samples using positive selection technologies,whereby centrifugation, selective binding to solid support orcombinations thereof have been used to capture target cells from asample. These ‘positive selection’ methods rely upon a characteristicsurface marker or ligand on the target cell to pull the target cell froma mixture by binding the surface marker or ligand to a selective bindingmember that is selective for it; typically the selective binding memberis affixed to a solid surface or particle, so that the target cell isreadily isolated by separating the remaining mixture from the solidsurface or particle. A comparison of the performance of a positiveselection method and the depletion approach described in the presentinvention is illustrated in Table 5.

TABLE 5 Classification Selection Depletion Control -1 1 1 Control -2 1 4Control -3 0 1 Control -4 1 3 Control -5 0 4 Control -6 0 4 Control -7 13 Control -8 0 2 Control -9 1 5 Spike1 -20 11 18 Spike2 -20 8 17 Spike3-20 11 27 Spike4 -20 25 17 Spike5 -50 39 56 Spike6 -50 84 54 Spike7 -100106 111 Spike8 -100 91 108 Spike9 -100 502 117 Patient-1 3 3 Patient-214 20 Patient-3 0 36 Patient-4 13 39 Patient-5 0 27 Patient-6 0 14Patient-7 0 13 Patient-8 0 18

In this study, the samples processed with the positive selection methodwere analyzed using magnetic beads coupled with anti-EpCAM monoclonalantibodies, to capture epithelial-like cells. In all the experimentsused to generate the data in Table 3 the blood samples were split intotwo aliquots which were processed in parallel using the two methods. Thefirst 9 samples were obtained from healthy control subjects. Thedepletion methods described in the present invention produced up to 5cancer-like cells while a slightly lower background was detected in thesamples processed using EpCAM-based selection (up to 1 cancer-like cellper sample).

To evaluate the general recovery rate of the two approaches bloodsamples from healthy individuals were spiked with 20, 50 or 100 tumorcells (as indicated in the “Classification”). In this experiment thedepletion method provided more consistent recovery efficiencies, betteraligned with the expected number of cancer cells in the sample. Thisdemonstrates that the negative selection method can perform better forthe purposes of detecting cancers, monitoring their treatment, etc. thanthe positive selection method. Recovery of the target cells would ofcourse be further reduced if some or all of the target cells hadundergone some type of modification of the surface marker or ligand(e.g., mutations) that the positive-selection method relied upon toaffix or label the target cells. Nevertheless, in some embodiments, thepresent methods may include a positive selection step as part of atarget cell isolation process, or as a labeling step to characterize thecells once they have been isolated or enriched.

This set of data was obtained from testing blood samples from patientspreviously diagnosed with breast cancer. For 1 patient (no. 1) there isgood agreement between the results obtained with the two methods. Fortwo other patients, number 2 and number 4, the depletion methods resultsin twice the number of cancer cells detected when compared to thepositive selection method. For all of the remaining patients however,the positive selection methods reported 0 cancer cells while thedepletion method allowed the detection of 13 to 36 cancer cells. Theseare patients known to have metastatic cancer for which the selectionmethod would have completely missed the diagnosis and for which thedepletion methods described in this patent provided a better diagnosticoption.

Example 7 Isolation of Rare Cells from Human Blood

In this example, blood samples from thirteen healthy donors were spikedwith different number of mitotracker/Hoechst-labeled SKBR or A549 cells.Five ml of human blood were mixed with the same volume of Hank'sbuffered saline containing 5 mM EDTA and 0.5% BSA, followed by adding0.1 ml of anti-CD50 conjugated magnetic beads (diameter ˜350 nm;concentration of 4×10⁹/ml). Reaction solution was mixed at roomtemperature for 5 mins, and then loaded on the top of 3 ml separationmedium based on Ficoll, diluted to 90% with Hank's buffer (For Ficoll,density=1.077 g/100 ml). The solution was centrifuged at 350 g for 5 minat room temperature. Supernatant above the RBC layer was collected,followed by spinning at 1200 g for 5 min at room temperature. The cellpellet was collected for microscopic analysis. The result is listed inTable 6.

TABLE 6 Exp't Cell # Spiked Cell # Recovered Recovery Rate 1 37 33 89% 243 38 88% 3 40 38 95% 5 60 52 87% 6 54 35 65% 7 47 36 77% 8 31 28 90% 942 33 79% 10 34 31 91% 11 44 31 70% 12 37 30 81% 13 36 32 89% Average83% SD  9%

Example 8 Detection of CTCs from Lung Cancer Patient Blood Samples andCorrelation with Anti-Cancer Therapy

Blood Collection: 7.5 ml venous blood sample was drawn into BDVacutainer® ACD tubes after the initial 2 ml was discarded. For CTC andCT scan correlation study, blood samples were drawn from 12 late stagelung cancer patients one day before and two weeks after cancer patientscompleted chemotherapy. WBCs were prepared and incubated with CD50antibody coated magnetic beads. After bead separation, enriched rarecell portion was centrifuged and the resulting cell pellet was suspendedand spotted on coated slides, and fixed. Cancer cell detection byimmunofluorescence staining Slides were stained with anti-cytokeratin8/18 Alexa 594, anti-cytokeratin 19 Alexa 488, and counterstained byDAPI and examined under a fluorescent microscope. Cancer progressionmonitoring Clinical responses were monitored by CT scan two months afterthe completion of chemotherapy and judged according to the RECIST. (FIG.6)

All publications, including patent documents and scientific articles,referred to in this application and the bibliography and attachments areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication were individually incorporatedby reference.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims. Citation of the above publications ordocuments is not intended as an admission that any of the foregoing ispertinent prior art, nor does it constitute any admission as to thecontents or date of these publications or documents.

1. A method of enriching target cells in a sample, which methodcomprises: a) Removing white blood cells (WBCs) from a biological sampleby allowing them to bind to a specific binding member affixed to a solidsupport; b) Removing red blood cells (RBCs) from the sample with adensity-based method, to produce a sample enriched in the target celltype; and c) Performing an analysis, manipulation or application stepwith the sample enriched in the target cell type to determine the numberor presence of target cells. 2-37. (canceled)